Methods of Producing Patient-Specific Anti-Cancer Therapeutics and Methods of Treatment Therefor

ABSTRACT

A method of preparing an antibody therapeutic is provided comprising: (a) providing a dissociated cell sample from at least one solid tumor sample obtained from a patient; (b) loading the dissociated cell sample into a microfluidic device having a flow region and at least one isolation region fluidically connected to the flow region; (c) moving at least one B cell from the dissociated cell sample into at least one isolation region in the microfluidic device, thereby obtaining at least one isolated B cell; and (d) using the microfluidic device to identify at least one B cell that produces antibodies capable of binding to cancer cells. The cancer cells can be the patient&#39;s own cancer cells. Also provided are methods of treating patients, methods of labeling or detecting cancer, engineered T or NK cells comprising antibodies or fragments thereof, and engineered antibody constructs.

This application is a continuation of U.S. patent application Ser. No.15/406,289, filed Jan. 13, 2017, which is a non-provisional applicationclaiming the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication No. 62/279,341, filed on Jan. 15, 2016; U.S. ProvisionalApplication No. 62/411,690, filed on Oct. 23, 2016; and of U.S.Provisional Application No. 62/412,092, filed on Oct. 24, 2016, each ofwhich disclosures is herein incorporated by reference in its entirety.

FIELD

Methods for isolating B cells that produce cancer-specific antibodiesare provided, as well as methods of treatment using those antibodiesand/or derivatives thereof.

BACKGROUND

Immunotherapy is the burgeoning field of using a patient's own immunesystem to help fight cancer. A variety of immunotherapy strategies havebeen evaluated, including stimulating the patient's own immune system toattack cancer cells or administering immune system components from anexternal source. For example, monoclonal antibodies designed to attackcancer cells in vivo have been administered alone or in geneticallyengineered constructs. In addition, CAR-T (chimeric antigen receptor Tcell) therapy has been investigated. In this therapeutic approach,genetically engineered T cells express antibody-containing fusionproteins on their surface, which target the T cells to the cancer inquestion and allows for the T cells to kill the cancer cells. Becausethe CAR-T cells can become permanently engrafted in the patient's body,this approach seems particularly promising. These approaches, however,still require further refinement.

One of the key problems in monoclonal antibody therapy or CAR-T therapyis identifying or designing an antibody that will provide the maximumbenefit to the patient in question, keeping in mind that the patient mayhave a very short window of time before the treatment can begin orbefore treatment success is required to prevent significant morbidityand mortality.

The present embodiments offer a solution to identifying antibodies thatwill provide the maximum benefit to the patient in question, minimizingthe amount of time required for research investigation and allowing fortreatment to begin as soon as possible.

SUMMARY

In accordance with the description, a method of preparing an antibodytherapeutic comprises:

-   -   a) providing a dissociated cell sample from at least one solid        tumor sample obtained from a patient;    -   b) loading the dissociated cell sample into a microfluidic        device having at least one isolation region;    -   c) moving at least one B cell from the dissociated cell sample        into at least one isolation region in the microfluidic device,        thereby obtaining at least one isolated B cell;    -   d) identifying at least one isolated B cell that produces        antibodies capable of binding to a cancer cell-associated        antigen.

This method and others described herein provide the advantage ofidentifying B cells that, in some embodiments, the patient's own bodyhas produced in order to target the type of cancer that the patient issuffering from. By using a microfluidic device to isolate theseantibodies, a plurality of B cells can be tested in parallel in arelatively rapid assay format and specific B cells of interest can beidentified, cultured, and sequenced (or used to produce a hybridoma).This enables investigators to produce patient-specific anti-cancerantibodies, which can be administered as monoclonal antibodies orfragments thereof, or genetically engineered constructs such as fusionproteins or CAR-T therapeutics, or used in methods of detection, etc.

Additional embodiments include methods of treating a patient havingcancer comprising treating the patient with an antibody or fragmentthereof produced by the methods herein. Cancer in a patient may also belabeled or detected using antibodies or fragments thereof conjugated toa detectable label. As compositions for treatments, engineered T cellscomprising the antibodies or fragments thereof displayed on theirsurface may be provided, as well as engineered antibody constructs thatcomprise at least the heavy chain CDRs of the antibody identifiedherein, at least the heavy and light chain CDRs, at least the heavychain variable region, or at least the heavy and light chain variableregions.

Additional objects and advantages will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice. The objects and advantageswill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) andtogether with the description, serve to explain the principles describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an example of a microfluidic device and a system foruse with the microfluidic device, including associated control equipmentaccording to some embodiments.

FIGS. 1B and 1C illustrate vertical and horizontal cross-sectionalviews, respectively, of a microfluidic device according to someembodiments.

FIGS. 2A and 2B illustrate vertical and horizontal cross-sectionalviews, respectively, of a microfluidic device having isolation pensaccording to some embodiments.

FIG. 2C illustrates a detailed horizontal cross-sectional view of asequestration chamber according to some embodiments.

FIG. 2D illustrates a partial horizontal cross-sectional view of amicrofluidic device having isolation pens according to some embodiments.

FIGS. 2E and 2F illustrate detailed horizontal cross-sectional views ofsequestration chambers according to some embodiments.

FIG. 2G illustrates a microfluidic device having a flow region whichcontains a plurality of flow channels, each flow channel fluidicallyconnected to a plurality of sequestration chambers, according to anembodiment.

FIG. 2H illustrates a partial vertical cross-sectional view of amicrofluidic device in which the inward facing surface of the base andthe inward facing surface of the cover are conditioned surfacesaccording to some embodiments.

FIG. 3A illustrates a specific example of a system nest, configured tooperatively couple with a microfluidic device, and associated controlequipment according to some embodiments.

FIG. 3B illustrates an optical train of a system for controlling amicrofluidic device according to some embodiments.

FIG. 4 illustrates steps in an exemplary workflow for identifying a Bcell lymphocyte expressing an antibody that specifically binds to acancer cell-associated antigen according to some embodiments.

FIGS. 5A-5C depict a microfluidic device comprising a plurality ofmicrofluidic channels, each fluidically connected with a plurality ofsequestration chambers. Each sequestration chamber contains a pluralityof mouse spenocytes. FIG. 5A is a bright field image of a portion of themicrochannel device. FIGS. 5B and 5C are fluorescence images obtainedusing a Texas Red filter. In FIG. 5B, the image was obtained 5 minutesafter the start of the antigen specificity assay described in Example 1.In FIG. 5C, the image was obtained 20 minutes after the start of theantigen specificity assay described in Example 1. The white arrows inFIG. 5C point to sequestration chambers that generated a positive signalin the assay.

FIG. 6 shows a Western blot of various fractions from Jurkat cells.

FIG. 7 shows beads coated with Jurkat cell membrane fractions stainedwith anti-CD3, anti-CD45 or anti-Na/K-ATPase antibodies.

FIG. 8 shows beads coated with HEK293 cells stained with the sameantibodies as in FIG. 7.

DETAILED DESCRIPTION OF THE EMBODIMENTS I. Definitions

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the figures may show simplified or partial views, and the dimensions ofelements in the figures may be exaggerated or otherwise not inproportion. In addition, as the terms “on,” “attached to,” “connectedto,” “coupled to,” or similar words are used herein, one element (e.g.,a material, a layer, a substrate, etc.) can be “on,” “attached to,”“connected to,” or “coupled to” another element regardless of whetherthe one element is directly on, attached to, connected to, or coupled tothe other element or there are one or more intervening elements betweenthe one element and the other element. Also, unless the context dictatesotherwise, directions (e.g., above, below, top, bottom, side, up, down,under, over, upper, lower, horizontal, vertical, “x,” “y,” “z,” etc.),if provided, are relative and provided solely by way of example and forease of illustration and discussion and not by way of limitation. Inaddition, where reference is made to a list of elements (e.g., elementsa, b, c), such reference is intended to include any one of the listedelements by itself, any combination of less than all of the listedelements, and/or a combination of all of the listed elements. Sectiondivisions in the specification are for ease of review only and do notlimit any combination of elements discussed.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

The term “ones” means more than one.

As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10,or more.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “microfluidic device” or “microfluidic apparatus” is adevice that includes one or more discrete microfluidic circuitsconfigured to hold a fluid, each microfluidic circuit comprised offluidically interconnected circuit elements, including but not limitedto region(s), flow path(s), channel(s), chamber(s), and/or pen(s), andat least two ports configured to allow the fluid (and, optionally,micro-objects suspended in the fluid) to flow into and/or out of themicrofluidic device. Typically, a microfluidic circuit of a microfluidicdevice will include at least one microfluidic channel and at least onechamber, and will hold a volume of fluid of less than about 1 mL, e.g.,less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9,8, 7, 6, 5, 4, 3, or 2 μL. In certain embodiments, the microfluidiccircuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15,2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150,20-200, 50-200, 50-250, or 50-300 μL.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element configured to hold a volume of fluid ofless than about 1 μL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. Ananofluidic device may comprise a plurality of circuit elements (e.g.,at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). Incertain embodiments, one or more (e.g., all) of the at least one circuitelements is configured to hold a volume of fluid of about 100 pL to 1nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g.,all) of the at least one circuit elements is configured to hold a volumeof fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL, 100 to400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL,or 250 to 750 nL.

A “microfluidic channel” or “flow channel” as used herein refers to aflow region of a microfluidic device having a length that issignificantly longer than both the horizontal and vertical dimensions.For example, the flow channel can be at least 5 times the length ofeither the horizontal or vertical dimension, e.g., at least 10 times thelength, at least 25 times the length, at least 100 times the length, atleast 200 times the length, at least 500 times the length, at least1,000 times the length, at least 5,000 times the length, or longer. Insome embodiments, the length of a flow channel is in the range of fromabout 50,000 microns to about 500,000 microns, including any rangetherebetween. In some embodiments, the horizontal dimension is in therange of from about 100 microns to about 1000 microns (e.g., about 150to about 500 microns) and the vertical dimension is in the range of fromabout 25 microns to about 200 microns, e.g., from about 40 to about 150microns. It is noted that a flow channel may have a variety of differentspatial configurations in a microfluidic device, and thus is notrestricted to a perfectly linear element. For example, a flow channelmay include one or more sections having any of the followingconfigurations: curve, bend, spiral, incline, decline, fork (e.g.,multiple different flow paths), and any combination thereof. Inaddition, a flow channel may have different cross-sectional areas alongits path, widening and constricting to provide a desired fluid flowtherein.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, a microfluidicsequestration chamber and a microfluidic channel, or a connection regionand an isolation region of a microfluidic sequestration chamber.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between a microfluidic sequestration chamberand a microfluidic channel, or at the interface between an isolationregion and a connection region of a microfluidic sequestration chamber.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be isolated and collected in accordance withthe present invention. Non-limiting examples of micro-objects include:inanimate micro-objects such as microparticles; microbeads (e.g.,polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells (e.g., embryos, oocytes, ova, sperm cells,cells dissociated from a tissue, eukaryotic cells, protist cells, animalcells, mammalian cells, human cells, immunological cells, hybridomas,cultured cells, cells from a cell line, cancer cells, infected cells,transfected and/or transformed cells, reporter cells, prokaryotic cells,and the like); biological organelles; vesicles, or complexes; syntheticvesicles; liposomes (e.g., synthetic or derived from membranepreparations); lipid nanorafts (as described in Ritchie et al. (2009)“Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,”Methods Enzymol., 464:211-231), and the like; or a combination ofinanimate micro-objects and biological micro-objects (e.g., microbeadsattached to cells, liposome-coated micro-beads, liposome-coated magneticbeads, or the like). Beads may further have other moieties/moleculescovalently or non-covalently attached, such as fluorescent labels,proteins, small molecule signaling moieties, antigens, orchemical/biological species capable of use in an assay.

As used herein, the term “cell” refers to a biological cell, which canbe a plant cell, an animal cell (e.g., a mammalian cell), a bacterialcell, a fungal cell, or the like. A mammalian cell can be, for example,from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, aprimate, or the like.

A colony of biological cells is “clonal” if all of the living cells inthe colony that are capable of reproducing are daughter cells derivedfrom a single parent cell. The term “clonal cells” refers to cells ofthe same clonal colony.

As used herein, “colony” of biological cells refers to 2 or more cells(e.g. 2-20, 4-40, 6-60, 8-80, 10-100, 20-200, 40-400, 60-600, 80-800,100-1000, or greater than 1000 cells).

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, averaged over time, is less than the rate of diffusion ofcomponents of a material (e.g., an analyte of interest) into or withinthe fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through thedevice.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

As used herein: μm means micrometer, μm3 means cubic micrometer, pLmeans picoliter, nL means nanoliter, and μL (or uL) means microliter.

Section divisions in the specification are for ease of review only anddo not limit any combination of elements discussed.

II. Method of Preparing an Antibody Therapeutic

An antibody therapeutic may be prepared using a method comprising thesteps of:

-   -   a) providing a dissociated cell sample from at least one solid        tumor sample obtained from a patient;    -   b) loading the dissociated cell sample into a microfluidic        device having a flow region and at least one isolation region        fluidically connected to the flow region;    -   c) moving at least one B cell from the dissociated cell sample        into at least one isolation region in the microfluidic device,        thereby obtaining at least one isolated B cell; and    -   d) identifying at least one isolated B cell that produces        antibodies capable of binding to a cancer cell-associated        antigen.

This method of identifying antibodies may be practiced in a variety ofdifferent modes, keeping in mind one potential goal of having arelationship between the B cells producing the antibodies and the cancercells that are posing the problem for the patient for whom a therapeuticis desired. One particular method 400 is outlined in FIG. 4.

In certain instances, the method further comprises determining pairedheavy chain and light chain variable domain antibody sequences from theidentified B cell(s). For example, the sequencing may be performed afterat least one B cell producing an antibody binding to the cancer cells isexported from the microfluidic device. In other instances, the methodcomprises generating a hybridoma from at least one isolated B cell. Ifthe antibody sequence is determined, the method may also comprisegenerating an antibody therapeutic comprising some or all of the pairedheavy chain and light chain variable domain sequences from the at leastone identified B cell. For example, a method may comprise preparing anantibody or functional part thereof that comprises all six CDRs from theheavy and light chain variable domain sequences from the at least oneidentified B cell.

A. Preparation of the Dissociated Cell Sample

In certain instances, the methods include the step of obtaining a samplefrom a solid tumor. For example, as shown in step 410 of method 400 inFIG. 4. The solid tumor sample can be a tumor biopsy, such as asurgically resected biopsy or a fine needle aspirate (FNA). In someinstances, the tumor has a tertiary lymphoid structure, which maycomprise proliferating B cells and/or a B-cell follicle. The tumor maybe breast cancer, genitourinary cancer, a cancer of the nervous system,intestinal cancer, lung cancer, melanoma, or another type of cancer. Insome embodiments, the breast cancer may be a medullary breast cancer. Insome embodiments, the genitourinary cancer may be a cancer originatingin the urinary tract, such as in the kidneys (e.g., renal cellcarcinoma), ureters, bladder, or urethra. In some embodiments, thegenitourinary cancer may be a cancer of the male reproductive tract(e.g., testicular cancer, prostate cancer, or a cancer of the seminalvesicles, seminal ducts, or penis) or of the female reproductive tract(e.g., ovarian cancer, uterine cancer, cervical cancer, vaginal cancer,or a cancer of the fallopian tubes). In some embodiments, the cancer ofthe nervous system may be neuroblastoma. In some embodiments, theintestinal cancer may be colorectal cancer. In some embodiments, thelung cancer may be mesothelioma.

In some instances, a single tumor sample is obtained. In otherinstances, multiple tumor samples are obtained. In one instance, alltumor samples are from the same patient. For example, one tumor samplemay be used from the patient (such as a single biopsy) or multiple tumorsamples may be used from the same patient (such as multiple biopsiesfrom the same tumor or from different tumors in the patient).

In other instances, tumor samples may be used from different patients.For example, the B cells can be from a first patient and cancer cells(e.g., cancer cells used as a source of cancer cell-associated antigen)are from a second patient. In some of these modes, the B cells are fromthe patient who desires treatment. In some of these modes the cancercells are from a cancer cell line. If the samples are from differentpatients, some relationship between the B cells producing the antibodiesand the cancer cell-associated antigen may also be preserved. Forexample, if the cancer cell-associated antigen is not from the cancercells of the same patient, in some embodiments, they may be from thesame type of cancer. In certain instances, cancer cells used in themicrofluidic device or otherwise used to provide cancer-associatedantigen exhibit one or more markers that are characteristic of the typeof tumor that the patient is suffering from. For example, the cancercells can have one or more such markers that are also present in thepatient's tumor sample.

In certain instances, the methods include the step of processing thetumor sample to produce a dissociated cell sample. For example, as shownin step 420 of method 400 in FIG. 4. The dissociated cell sample maycomprise at least one single cell that is dissociated from a tumor(e.g., tumor biopsy or FNA). In some instances, all of the cells are insingle cell form. In other instances, some of the cells are not insingle cell form, but remain in clumps of about 2, 3, 4, 5, 6, 7, 8, 9,10 cells or more, yet other cells are in single cell form. A dissociatedcell sample includes a cancer cell line grown in culture in dissociatedform. Thus, the cells may either be actively dissociated (by obtainingat least one solid tumor sample and dissociating at least one singlecell) or passively dissociated (by obtaining a sample that has beenpreviously dissociated or grown in dissociated form).

The dissociation may be conducted in a number of ways. For example, thetumor sample may be dissociated using a collagenase plus DNasedigestion. The tumor sample may also be dissociated using a celldissociator instrument, such as the gentleMACS™ instrument from MiltenyiBiotec.

In some instances, the method further comprises performing a selectionon the dissociated cell sample prior to loading, to isolate a fractionthat has a greater percentage and/or concentration of B cells than theoriginal dissociated sample. For example, as shown in step 430 of method400 in FIG. 4. If desired, B cells may be selected from the dissociatedcell sample using at least one marker, such as those chosen from CD19,CD20, IgM, IgD, CD38, CD27, CD138, PNA, and GL7. Alternatively, or inaddition, a negative selection may be performed to remove non-B cells(e.g., using at least one marker that is not expressed on B cells). Forexample, the dissociated cell sample may be depleted of cancer cells(e.g., using a cancer-cell specific marker) and/or T cells (e.g., usinga T cell-specific marker, such as CD3, CD4, CD8, etc.). In still otherinstances, the dissociated cell sample is loaded into the microfluidicdevice without being processed to enrich for B cells.

Regardless of whether the dissociated cell sample is processed to enrichfor B cells, the dissociated cell sample is loaded into a microfluidicdevice. See, for example, step 440 of method 400 in FIG. 4. In someinstances, a single dissociated cell sample may be loaded onto themicrofluidic device. The single dissociated cell sample may compriseboth B cells and other types of cells (e.g., cancer cells). In otherinstances, (i) separate fractions of a single dissociated cell sample or(ii) different dissociated cell samples (e.g., each fractionatedseparately and/or in a different manner), may be loaded at differenttimes. In some instances, a dissociated cell sample may be processed toproduce a fraction that has a greater percentage and/or concentration ofcancer cells than the original sample. The cancer cell fraction may bethe fraction that remains after selection of B cells, or vice versa.

If desired, the cancer cells may be selected from the dissociated cellsample using at least one marker characteristic of the cancer. In someinstances, the cells may be separated using morphological differences.Cancer cells often display irregular cell size (e.g., larger cell size,or smaller cell cell), bigger nuclei, contain more DNA, or containnuclear structural changes. In many cases, these differences can bediscerned visually and/or by image analysis, which may be automated. Tofacilitate or enhance such analysis, the dissociated cell sample can bestained for nucleic acids (e.g., when stained with a nucleicacid-binding dye, cancer cells often stain brighter than normal cells)or for markers associated with the nuclear envelope, nucleoli, and/ornuclear matrix. Examples of such markers include lamins (e.g., A- orB-type lamins), nuclear membrane proteins (e.g., nuclearlamina-associated proteins, such as emerin), fibrillarin, nuclear poreproteins (NUPs, such as Nup153, Nup210, etc.), histone proteins, andnuclear matrix proteins (e.g., p84)), any of which can highlightdifferences in nuclear structure.

Aside from morphological differences, a variety of markers are known inthe art as being useful for identifying certain types of cancer cells,including but not limited to:

For breast cancer, CD44, HLA-DR, Ki-67 (or MK167), aldehydedehydrogenase 1 (ALDH1), and ganglioside GD2 tend to be present and/orelevated in cancer cells, while estrogen receptor (ER), progesteronereceptor (PR), human epidermal growth factor receptor 2 (HER2), and CD24tend to be absent or reduced in cancer calls; ER−/PR−/HER2−/HLA-DR⁺ canbe used to identify medullary breast cancer; andCD44^(hi)/CD24^(lo)/ALDH1^(hi) or CD44^(hi)/CD24^(lo)/GD2^(hi) can beused to identify breast cancer stem cells.

For renal cancer, C-reactive protein, aquaporin-1 (AQP1), adipophilin(ADFP), insulin-like growth factor II mRNA binding protein 3 (IGF2BP3 orIMP3), B7-H1 (or PD-L1), and Ki-67 (MK167) tend to be present and/orelevated in cancer cells.

For bladder cancer, nuclear mitotic apparatus protein (NMP22), bladdertumor antigen (BTA), and fibrin/fibrinogen degradation products tend tobe present and/or elevated in cancer cells, while adipocyte fatty acidbinding protein (A-FABP), glutathione S-transferase mu (GST-p),prostaglandin dehydrogenase (PGDH), and keratin 13 tend to be absent orreduced relative to normal cells; at the genetic level, the p16 tumorsuppressor gene or the 9p21 locus may be deleted in cancer cells.

For urothelial cancer, complement factor H-related protein (CFHrp) mayexhibit increased levels and/or secretion in cancer cells; at thenuclear level, telomerase reverse transcriptase (TERT) tends to exhibitincreased mRNA expression in cancer cells.

For endometrial cancer, cancer antigen 15-3 (CA15-3), cancer antigen 125(CA125), cancer antigen 19.9 (CA19.9), cancer antigen 72.4 (CA72.4), andcarcinoembryonic antigen (CEA) tend to be present and/or elevated incancer cells.

For ovarian cancer, tumor-associated trypsin inhibitor (TATI), cancerantigen 125 (CA125), Claudin5, human epidydmis protein 4 (HE4),carcinoembryonic antigen (CEA), VCAM-1, and miR-181a tend to be presentand/or elevated in cancer cells; TATI, CA125, and Claudin 5 have beenused in combination to diagnose ovarian cancer, as have HE4 and CA125,optionally in conjunction with CEA and VCAM-1; STAT1 can be used todistinguish between ovarian cancers that are responsive to chemotherapyand those that are not.

For cervical cancer, human papilloma virus (HPV) (e.g., types 16, 18,31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 6, 11, 42, 43, and 44),p16^(INK4a), and insulin-like growth factor II mRNA binding protein 3(IGF2BP3 or IMP3) tend to be present and/or elevated in cancer cells.

For prostate cancer, prostate specific antigen (PSA), sarcosine (ametabolite), prostate cancer gene 3 (PCA3), and TMPRSS2:ERG fusionproduct tend to be present and/or elevated in cancer cells, whileprostatic acid phosphatase, fibrinogen a chain precursor, collagen α-1(III), collagen α-1 (I), psoriasis susceptibility 1 candidate gene 2protein, hepatocellular carcinoma associated protein TB6, histone H2BB,osteopontin, polymeric Ig receptor, Na/K-transporting ATPase γ,transmembrane secretory component, and semenogelin 1 tend to be absentor reduced relative to normal cells.

For neuroblastoma, increased levels and/or secretion of vanillylmandelicacid (VMA), homovanillic acid (HVA), and ferritin are associated withcancer cells, and neuron-specific enolase (NSE), lactate dehydrogenase(LDH), and ganglioside GD2 tend to be present and/or elevated in cancercells; at the genomic level, deletions in parts of chromosomes 1p and11q and duplication of a part of 17q are associated with cancer cells.

For colorectal cancer, carcinoembryonic antigen (CEA), cancer antigen19-9 (CA19-9), colon-cancer-specific antigen 3 (CCSA-3),colon-cancer-specific antigen 4 (CCSA-4), and B-Raf mutation V600E tendto be present and/or elevated in cancer cells; at the genetic level,colorectal cancer cells exhibit microsatellite instability and variousK-Ras mutations.

For small cell lung carcinoma, ganglioside GD2 tends to be presentand/or elevated in cancer cells; for non-small cell lung carcinoma,B-Raf mutation V600E tends to be present in cancer cells; formesothelioma, calretinin, cytokeratin 5/6, and WT1 tend to be presentand/or elevated in cancer cells, while carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (Ep-CAM)(e.g., as detected by theMOC-31 or Ber-EP4 antibodies), Lewis Y blood group (e.g., as detected bythe BG-8 antibody), and the tumor associated glycoprotein detected bythe B72.3 antibody tend to be absent or reduced; and, conversely, forpulmonary adenocarcinoma, CEA, Ep-CAM (e.g., as detected by the MOC-31or Ber-EP4 antibodies), Lewis Y blood group (e.g., as detected by theBG-8 antibody), and the tumor associated glycoprotein detected by theB72.3 antibody tend to be present and/or elevated in cancer cells, whilecalretinin, cytokeratin 5/6, and WT1 tend to be absent or reduced.

For melanoma, the human endogenous retrovirus (HERV-K), ganglioside GD2,B-Raf mutation V600E, Hsp90, regulator of G-protein signaling 1 (RGS1),Osteopontin, human epidermal growth factor receptor 3 (HER3), nuclearreceptor coactivator 3 (NCOA3), and minichromosome maintenance complexcomponents 4 and 6 (MCM4 and MCM6, respectively) tend to be presentand/or elevated in cancer cells, while inhibitor or growth proteins 3and 4 (ING3 and ING4, respectively) tend to be absent or reducedrelative to normal cells.

In some instances, the cancer cells may be selected from the dissociatedcell sample using at least two markers characteristic of the cancer. Forexample, the two or more markers can include a morphological marker, atleast one positive marker (e.g., a marker which is present and/orelevated in the cancer cells), and/or at least one negative marker(e.g., a marker which is absent or reduced relative to normal cells), orany combination thereof. In certain modes, the B cells (e.g., memory Bcells, plasma B cells, or the like, and combinations thereof) and/orcancer cells may be selected from the dissociated cell sample by FACS.Alternatively, or in addition, the B cells (e.g., memory B cells, plasmaB cells, or the like, and combinations thereof) and/or cancer cells mayalso be selected from the dissociated sample by using magneticbead-based sorting. The selection can enrich the sample for B cells thatexpress CD27 (or some other memory B cell marker) or for B cells thatexpress CD138 (or some other plasma cell marker). The selection can bepositive (e.g., based on a B cell-specific or a cancer cell-specificmarker). Alternatively, the selection can be negative. For example,non-B cell cell types can be depleted from the dissociated cell sampleusing techniques that are well known in the art, such as treating thesample with the DYNABEADS™ Untouched Human B Cells reagent (ThermoFisher), the B Cell Isolation Kit (Miltenyi), the RosetteSep Human BCell Enrichment Cocktain (Stem Cell Technologies), or the like. Asanother example, the selection can deplete the sample of B cellsexpressing IgM antibodies, IgA antibodies, IgD antibodies, IgGantibodies, or any combination thereof. Alternatively, or in addition,the B cells and/or cancer cells may be selected using a microfluidicdevice, as discussed further below.

B. Loading and Moving B Cells into and within the Microfluidic Device

The dissociated cell sample (optionally fractionated as discussed above)can be loaded into the microfluidic device by flowing the cells of thesample through an inlet in the device and into the flow region. Forexample, step 440 of method 400 shown in FIG. 4 provides for loading thedissociated cell sample into the microfluidic device. In someembodiments, the flow region includes a flow channel (or microfluidicchannel), and loading the dissociated cell sample includes flowing thecells into the flow channel.

Once the dissociated cell sample is loaded into the flow region (or flowchannel) of the microfluidic device, B cells in the sample can be movedinto isolation regions of the microfluidic device. For example, asillustrated in step 450 of method 400 (FIG. 4), B cells can be movedfrom the flow region (or flow channel) into one or more isolationregions, which may be located within isolation chambers that arefluidically connected with and open off of the flow region (or flowchannel). The B cell can be, for example, a CD27⁺ B cell or a CD138⁺ Bcell. In some embodiments, the B cell is a memory B cell. In otherembodiments, the B cell is a plasma cell. The movement of the B cellsmay be accomplished by a variety of means, as discussed generallyherein, including using gravity (such as by tipping the microfluidicdevice), inducing localized fluid flow (such as by depressing or pullinga deformable surface of the microfluidic device located adjacent orproximal to the isolation region), applying dielectrophoretic (DEP)force (such as by optoelectronic tweezers (OET)), or any combinationthereof.

In some instances, prior to loading the dissociated cell sample, themethod further comprises labeling B cells in the dissociated cell samplewith at least one detectable marker. The marker may be a B cell-specificmarker, such as any one of the B cell markers disclosed herein (e.g.,CD19, CD20, IgM, IgD, CD38, CD27, CD138, PNA, GL7, and the like). Themarker may aid in selecting B cells for movement based on the detectionof the detectable marker, such as in the embodiment where the B cell ismoved by applying DEP force. Alternatively, the detectable marker canbind to a non-B cell marker, such as a T cell marker or a cancercell-associated marker. In such instances, B cells can be selected formovement based on their lack of the detectable marker.

The B cells loaded into the isolation region(s) of the microfluidicdevice can include a mixture of differ B cell types, such as memory Bcells, plasma cells, and the like. In some embodiments, it may bedesirable to select plasma cells for movement into the isolationregions. In some embodiments, it may be desirable to select B cell thatexpress an IgG-type antibody for movement into the isolation regions.

In some instances, only one B cell is moved into each isolation region,whether all of the isolation regions are loaded or not. In otherinstances, the B cells can be initially pooled into one or moreisolation regions, and later separated into individual isolation regions(i.e., one B cell per isolation region). The latter embodiments allowfor greater throughput when screening the B cells for production ofantibodies that bind to the cancer cells. Placement of only one B cellin each isolation region allows for clonal expansion of the B cell,which can facilitate the identification of paired heavy chain and lightchain variable domain sequences.

In certain embodiments, the B cells are loaded into the microfluidicdevice and moved into the isolation regions before the cancercell-associated antigen is loaded into the microfluidic device. In otherembodiments, the B cells are loaded into the microfluidic device andmoved into the isolation regions after the cancer cell-associatedantigen have been loaded into the microfluidic device and moved into theisolation regions. In still other embodiments, the B cells and thecancer cell-associated antigen are loaded into the microfluidic deviceat the same time (e.g., as part of a dissociated cell sample thatincludes both B cells and cancer cells). In some embodiments, once the Bcells are loaded into the isolation regions they remain there untilafter at least one B cell produces antibodies capable of binding to thecancer cell-associated antigen has been identified.

C. Contacting B Cells that Produce Antibodies with CancerCell-Associated Antigens

The disclosed methods include the step of detecting whether a B cell isexpressing an antibody that specifically binds to a cancercell-associated antigen. See, for example, step 470 of method 400 inFIG. 4. To detect such expression, the expressed antibody must beallowed to interact with the cancer cell-associated antigen.

The cancer cell-associated antigen can be simple or complex; the antigencan be an epitope on a protein, a carbohydrate group or chain, abiological or chemical agent other than a protein or carbohydrate, orany combination thereof; the epitope may be linear or conformational.The cancer cell-associated antigen can be an antigen that uniquelyidentifies cancer cells (e.g., one or more particular types of cancercells) or is upregulated on cancer cells as compared to its expressionon normal cells. Typically, the cancer cell-associated antigen ispresent on the surface of the cancer cell, thus ensuring that it can berecognized by an antibody. The antigen can be associated with any typeof cancer cell, including any type of cancer cell that can be found in atumor known in the art or described herein. In particular, the antigencan be associated with lung cancer, breast cancer, melanoma, and thelike. As used herein, the term “associated with a cancer cells,” whenused in reference to an antigen, means that the antigen is produceddirectly by the cancer cell or results from an interaction between thecancer cell and normal cells.

Detecting whether a B cell expressed antibody specifically binds to acancer cell-associated antigen can be performed in a microfluidic devicedescribed herein. In particular, the microfluidic device can include anenclosure having a flow region (e.g., a microfluidic channel) and asequestration chamber. The sequestration chamber can include anisolation region and a connection region, the connection regionproviding a fluidic connection between the isolation region and the flowregion. The sequestration chamber can have a volume of about 0.5 nL toabout 5.0 nl, or any range therein (e.g., about 0.5 nl to about 1.0 nl,about 0.5 nl to about 1.5 nl, about 0.5 nl to about 2.0 nl, about 1.0 nlto about 1.5 nl, about 1.0 nl to about 2.0 nl, about 1.0 nl to about 2.5nl, about 1.5 nl to about 2.0 nl, about 1.5 nl to about 2.5 nl, about1.5 nl to about 3.0 nl, about 2.0 nl to about 2.5 nl, about 2.0 nl toabout 3.0 nl, about 2.0 nl to about 3.5 nl, about 2.5 nl to about 3.0nl, about 2.5 nl to about 3.5 nl, about 2.5 nl to about 4.0 nl, about3.0 nl to about 3.5 nl, about 3.0 nl to about 4.0 nl, about 3.0 nl toabout 4.5 nl, about 3.5 nl to about 4.0 nl, about 3.5 nl to about 4.5nl, about 3.5 nl to about 5.0 nl, about 4.0 nl to about 4.5 nl, about4.0 nl to about 5.0 nl, about 4.5 nl to about 5.0 nl, or any rangedefined by one of the foregoing endpoints). The connection region canhave a width W_(con) as generally described herein (e.g., about 20microns to about 100 microns, or about 30 microns to about 60 microns).The isolation region can likewise have a width W_(iso) that is generallyas described herein (e.g., the isolation region can have a width W_(iso)that is greater than the width W_(con) of the connection region). Incertain embodiments, the isolation region has a width W_(iso) that isabout 50 microns to about 250 microns.

The flow region, the sequestration chamber, and/or the isolation regionof the sequestration chamber can include at least one surface coatedwith a coating material that promotes the viability of theantibody-expressing B cell (e.g., a memory B cell or a plasma cell). Asused in this context, “promote the viability” means that the viabilityof the B cell is better on the coated surface as compared to anequivalent surface that is non-coated. In certain embodiments, the flowregion, the sequestration chamber, and/or the isolation region has aplurality of surfaces each coated with a coating material that promotesthe viability of the B cell. The coating material can be any suitablecoating material known in the art and/or described herein. The coatingmaterial can, for example, comprise hydrophilic molecules. Thehydrophilic molecules can be selected from the group of polymerscomprising polyethylene glycol (PEG), polymers comprising carbohydrategroups, polymers comprising amino acids, and combinations thereof.

The flow region, the sequestration chamber, and or the isolation regionof the sequestration chamber can include at least one conditionedsurface that promotes the viability of the B cell (e.g., a memory B cellor a plasma cell). As used in this context, “promote the viability”means that the viability of the B cell is better on the conditionedsurface as compared to an equivalent surface that is not conditioned. Incertain embodiments, the flow region, the sequestration chamber, and/orthe isolation region has a plurality of conditioned surfaces each ofwhich is capable of promoting the viability of the B cell. Theconditioned surface(s) can comprise covalently linked molecules. Thecovalently linked molecules can be any suitable molecules known in theart and/or disclosed herein, including, for example, covalently linkedhydrophilic molecules. The hydrophilic molecules can be selected fromthe group of polymers comprising polyethylene glycol (PEG), polymerscomprising carbohydrate groups, polymers comprising amino acids, andcombinations thereof. The hydrophilic molecules can form a layer ofcovalently linked hydrophilic molecules, as described herein.

Detecting a B cell expressing an antibody that specifically binds to acancer cell-associated antigen can include introducing the cancercell-associated antigen into the microfluidic device such that theantigen becomes located proximal to the B cell(s). Introducing thecancer cell-associated antigen into the microfluidic device can include,for example, flowing a fluidic medium that contains the cancercell-associated antigen into the flow region of the microfluidic deviceand stopping the flow of the fluidic medium when the cancercell-associated antigen is located proximal to the B cell(s). A location“proximal” to the B cell can be within 1 millimeter (mm) of the B cell(e.g., within 750 microns, within 600 microns, within 500 microns,within 400 microns, within 300 microns, within 200 microns, within 100microns, or within 50 microns of the B cell).

The cancer cell-associated antigen can be provided as part of amicro-object, which may be any suitable micro-object known in the artand/or described herein (e.g., a cell, a liposome, a lipid nanoraft, ora bead). Thus, introducing the cancer cell-associated antigen into themicrofluidic device can include positioning such a micro-object adjacentto or within the connection region of the sequestration chamber in whichthe B cell is located. Alternatively, introducing the cancercell-associated antigen can include moving such a micro-object into theisolation region of the sequestration chamber in which the B call islocated.

The micro-object can comprise antigen that has been fractionated fromcancer cells, such as membrane-associated antigens found in a cancercell membrane preparation. Such isolated membrane-associated antigensmay be conjugated to beads to produce the micro-objects used in thedisclosed methods. Alternatively, the micro-object can comprise asubstantially pure antigen (e.g., a purified protein). Methods ofconjugating antigenic molecules, such as purified proteins, cellmembrane preparations, and the like, to beads are known in the artand/or are described in the Examples herein. Likewise, methods ofisolating antigens from cancer cells are known in the art (see, e.g.,Bachleitner-Hoffmann et al. (2002), J. Clin. Endocrin. & Metab. 87(3):1098-1104; and He et al. (2016), Oncology Letters 12:1101-06). Thus, theisolation of cancer cell-associated antigens could be performed by avariety of methods known in the art. In other embodiments, themicro-object can be a cell, such as a cancer cell (e.g., isolated fromthe patient's sample) or a cell of a cancer cell line.

As indicated above, the micro-objects (e.g., cancer cells orantigen-conjugated beads) can be loaded into the microfluidic device(for the purpose of detecting antibody binding) by flowing themicro-objects through an inlet in the device and into the flow region.In some embodiments, the flow region includes a flow channel, andloading the micro-objects includes flowing the micro-objects into theflow channel.

In some embodiments, the micro-objects are loaded into the flow region(or flow channel) of the microfluidic device and remain in the flowregion (or flow channel) until exported from the microfluidic device. Insome embodiments, the micro-objects do not enter the isolation regions.In some embodiments, the micro-objects dip into the connection regionsof isolation chambers in addition to residing in the flow region (orflow channel). In any of these embodiments, the micro-objects (e.g.,cancer cells or antigen-conjugated beads) can be loaded into the flowregion (or flow channel) at a concentration of at least about 1×10⁷,2.5×10⁷, 5×10⁷, 7.5×10⁷, or 1×10⁸ micro-objects/ml.

In some embodiments, the micro-objects are moved into at least oneisolation region in the microfluidic device. As with the B cells,movement of the micro-objects (e.g., cancer cells or antigen-conjugatedbeads) can be accomplished by a variety of means, including usinggravity (such as by tipping the microfluidic device), inducing localizedfluid flow (such as by depressing or pulling a deformable surface of themicrofluidic device located adjacent or proximal to the isolationregion), applying dielectrophoretic (DEP) force (such as byoptoelectronic tweezers (OET)), or any combination thereof. In suchembodiments, one or more (e.g., only 1, about 2, 3, 4, 5, 6, 7, 8, 9,10, or more, or from about 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1 to 2)micro-objects can be moved into individual isolation regions in themicrofluidic device, thereby obtaining at least one isolation regionhaving an isolated micro-object or a group of isolated micro-objects.The isolated micro-object(s) and the B cell(s) may be placed in the sameisolation region(s). Alternatively, the isolated micro-object(s) and theB cell(s) may be placed in different isolation regions (such as inadjacent isolation regions).

Irrespective of the desired location for the micro-objects in themicrofluidic device, micro-objects that are cancer cells may be labeledwith one or more detectable markers prior to loading the cancer cellsinto the microfluidic device. If the desired mode includes moving atleast one cancer cell into at least one isolation region, the detectablemarker(s) may be used to select at least one cancer cell for movement.Alternatively, or in addition, morphological assessments of the cellsize, cell shape, nuclear size, or nuclear structure may be used toselect at least one cancer cell for movement. Cancer cells may also bechosen by the absence of a cellular marker, optionally in combinationwith morphological assessments.

In certain embodiments, the cancer cells originate from a dissociatedcell sample obtained from one or more solid tumor samples taken from thepatient, and thus are the patient's own cancer cells. As discussedabove, the cancer cells can be selected (or fractionated) from thedissociated cell sample and/or the cancer cells can be loaded into themicrofluidic device as part of the same dissociated cell sample thatcontains the B cells. In certain embodiments, the cancer cells arecultured and/or cloned prior to assaying the B cells for production ofantibodies capable of binding to the cancer cells. Such culturing and/orcloning can be performed within the microfluidic device or prior toloading the cancer cells into the microfluidic device (e.g., usingconventional techniques for selecting, culturing, and/or cloning cancercells from a tumor sample). Regardless, the cancer cells can beselected, cultured, and/or cloned to a concentration of at least about1×10⁷, 2.5×10⁷, 5×10⁷, 7.5×10⁷, or 1×10⁸ cells/ml.

As is well known in the art, a population of cancer cells derived from atumor can be relatively heterogeneous with respect to the morphologicaland genetic characteristics of individual cells that make up thepopulation. For example, the population may contain cancer stem cells(which may divide slowly) and more differentiated cancer cells (whichmay divide more rapidly and may contain differing subset of pro-cancermutations). Marker-based selection and/or cloning of cancer cells can beused to provide more homogeneous populations of cells. Accordingly, incertain embodiments, in some embodiments, a plurality of heterogeneouscancer cells may be loaded onto the microfluidic device. Alternatively,individual cancer cells may be selected and cloned before loading ontothe microfluidic device. Thus, in other embodiments, a substantiallyhomogeneous population of cancer cells can be loaded into themicrofluidic device and used to identify B cells that produce antibodiescapable of binding to the cancer cells. The substantially homogeneouspopulation can be a cancer cell line derived from the patient providingthe at least one tumor or from a different patient.

In still other embodiments, providing the cancer cell-associate antigencan involve flowing a solution comprising soluble antigen through theflow region of the microfluidic device and allowing the soluble antigento diffuse into the sequestration chamber in which the B cell islocated. Such soluble antigen can be covalently bound to a detectablelabel (e.g., a fluorescent label).

D. Detection of Binding and Processing of B Cells

Binding of antibodies produced by the B cells to the cancercell-associated antigen may be detected in a variety of ways. Forexample, when cancer cells are introduced into the microfluidic device,cell clumping may be detected, such as may occur in an agglutinationassay. Alternatively, a secondary antibody, such as an anti-humanantibody conjugated to a label (e.g., a fluorescent label), may be addedin order to label micro-objects (e.g., cancer cells orantigen-conjugated beads) that have antibody bound to them. Thus, insome embodiments, the methods further comprise providing a labeledantibody-binding agent prior to or concurrently with the cancercell-associated antigen. In such embodiments, monitoring of binding ofthe antigen antibody expressed by a B cell can involve detectingindirect binding of the labeled antibody-binding agent to the cancercell-associated antigen. The labeled antibody-binding agent can be alabeled anti-IgG antibody, which may be fluorescently labeled. Incertain embodiments, the labeled antibody-binding agent is provided in amixture with the cancer cell-assoiciated antigen. In other embodiments,the labeled antibody-binding agent is provided after providing thecancer cell-associated antigen.

In certain embodiments, the methods can further comprise identifying atleast one antibody expressing B cell (e.g., plasma cell or memory Bcell) as expressing an antibody that specifically binds to the cancercell-associated antigen. As discussed in more detail below, themicrofluidic device may comprise an imaging device that allows forvisualization of the signal (e.g., cancer cell clumping or accumulationof label at the surface of the micro-objects). For example, the imagingdevice can periodically image the microfluidic device and any increasesin such signal over time can be detected. The images can be obtain, forexample, every few seconds (e.g., every 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60 seconds, or more) or every few minutes (e.g., every 2, 3,4, 5, 6, 7, 8, 9, 10 minutes, or more). In some embodiments, a pluralityof images can be overlaid on top of one another, thereby creating a“summed” image which has the general effect of averaging out backgroundsignal and creating better contrast between real signal and backgroundsignal. The detection of signal can be manual, such as occurs when aperson reviews the images, or automated (e.g., using appropriate imageanalysis software).

Once binding has been detected between cancer cell-associated antigenand the antibodies produced by the B cells, B cells producing suchantibodies can be identified and their positions (e.g., the isolationregions or isolation chambers in which they are located) can be notedand/or recorded. See, for example, step 480 in method 400 of FIG. 4.Optionally, the flow region (or flow channel) may be flushed onceantibody binding to cancer cells has been detected. Such flushing mayoccur before or after the identification of B cells producing antibodiesthat bind to the cancer cell-associated antigen (e.g., cancer cells orantigen-conjugated beads). As another optional step, the B cells thatdid not produce antibodies capable of binding the cancer cell-associatedantigen may be discarded from the microfluidic device.

In certain embodiments, B cells identified as producing antibodies thatbind to the cancer cell-associated antigen can be exported from themicrofluidic device. For example, in order to isolate B cells for eithersequencing (e.g., of antibody heavy and light chain variable regions) orpreparation of a hybridoma, the B cells producing antibodies that bindto the cancer cells may be exported from the microfluidic device. Insome instances, each cell or population of cloned cells is exportedindividually.

E. Stimulation of B Cell Activation

In any of the foregoing embodiments, B cells that have been moved intoisolation regions in the microfluidic device can be incubated underconditions conducive to the production of antibodies. In some instances,those antibodies can diffuse through medium in the microfluidic deviceto the location of the cancer cell-associated antigen (whether comprisedby a cancer cell or antigen-conjugated bead, or in solution; and whetherin the same isolation region, the main flow channel, or in an adjacentisolation region). In the instance of adjacent isolation regions, insome microfluidic devices there exist small gaps in a thin wall betweentwo adjacent isolation regions, allowing the antibodies to diffusedirectly from one isolation region into another and without necessarilyneeding to enter the main flow channel to diffuse into an adjacentisolation region. Such small gaps allow for diffusion of antibodies butdo not allow for either the cancer cells or the B cells to leave theisolation regions.

In some instances, the detection of B cells that express an antibodythat specifically binds to a cancer cell-associated antigen can includethe step of contacting a B cell with a stimulating agent that stimulatesB cell activation. See, for example, step 460 in method 400 of FIG. 4.The stimulating agent can be a CD40 agonist, such as CD40L, a derivativethereof, or an anti-CD40 antibody. Thus, the stimulating agent cancomprise, consist essentially of, or consist of CD40L+ feeder cells. TheCD40L+ feeder cells can be T cells (e.g., Jurkat D1.1 cells), or aderivative thereof. Alternatively, the feeder cells can be a cell line(e.g., NIH-3T3 cells) transfected/transformed with a CD40L-expressingconstruct. The stimulating agent further comprises a toll-like receptor(TLR) agonist (e.g., a TLR9 agonist). The TLR agonist can be, forexample, a CpG oligonucleotide (e.g., CpG2006). The CpG oligonucleotidecan be used at a concentration of about 1 microgram/mL to about 20micrograms/mL (e.g., about 1.5 to about 15 micrograms/mL, about 2.0 toabout 10 micrograms/mL, or about 2.5 to about 5.0 micrograms/mL). The Bcell can be contacted (e.g., substantially continuously, orperiodically/intermittently) with the stimulating agent for a period ofone to ten days (e.g., two to eight days, three to seven days, or fourto six days).

Detecting a B cell expressing an antibody that specifically binds to acancer cell-associated antigen can further include the step of providingthe B cell with culture medium comprises one or more growth-inducingagents that promote B cell expansion. The one or more growth-inducingagents can include at least one agent selected from the group of IL-2,IL-4, IL-6, IL-10, IL-21, and BAFF. The IL-2 and/or IL-4 can be providedat a concentration of about 10 ng/mL to about 1 microgram/mL. The IL-6,IL-10, and/or IL-21 can be provided at a concentration of about 10 ng/mLto about 100 ng/mL The BAFF can be provided at a concentration of about10 ng/mL to about 50 ng/mL. In certain embodiments, the culture mediumis provided to the B cell for a period of one to ten days (e.g., two toeight days, three to seven days, or four to six days). The culturemedium can comprise the stimulating agent (e.g., CD40 agonist and/or TLRagonist). Thus, for example, providing the culture medium to the B cellcan be performed at the same time as contacting the B cell with theactivating agent. In certain embodiments, the steps of contacting the Bcell lymphocyte with a stimulating agent and providing culture medium tothe B cell lymphocyte are preformed at overlapping times (e.g., over asubstantially coextensive period of time). Additionally, as anotheroptional step, the B cells may be cultured into clonal populations inthe microfluidic device. Such culturing may occur, for example, forabout 2 to 3 days and/or to a cell count of about 8 to 20 cells.

III. Microfluidic Devices

A. Microfluidic Device Terms

In some embodiments, a microfluidic device can comprise “swept” regionsand “unswept” regions. An unswept region can be fluidically connected toa swept region, provided the fluidic connections are structured toenable diffusion but substantially no flow of media between the sweptregion and the unswept region. The microfluidic device can thus bestructured to substantially isolate an unswept region from a flow ofmedium in a swept region, while enabling substantially only diffusivefluidic communication between the swept region and the unswept region.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials (e.g., proteins, such asantibodies) can be assayed in such a microfluidic device. For example,sample material comprising biological micro-objects (such as a B cell)to be assayed for production of an analyte of interest (such as anantibody) can be loaded into a swept region of the microfluidic device.Ones of the biological micro-objects can be selected for particularcharacteristics and disposed in unswept regions. The remaining samplematerial can then be flowed out of the swept region and an assaymaterial flowed into the swept region. Because the selected biologicalmicro-objects are in unswept regions, the selected biologicalmicro-objects are not substantially affected by the flowing out of theremaining sample material or the flowing in of the assay material. Theselected biological micro-objects can be allowed to produce the analyteof interest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

B. System Including a Microfluidic Device

FIG. 1A illustrates an example of a microfluidic device 100 and a system150 which can be used for isolating and screening tumor-derived B cellsin vitro. A perspective view of the microfluidic device 100 is shownhaving a partial cut-away of its cover 110 to provide a partial viewinto the microfluidic device 100. The microfluidic device 100 generallycomprises a microfluidic circuit 120 comprising a flow path 106 throughwhich a fluidic medium 180 can flow, optionally carrying one or moremicro-objects (not shown) into and/or through the microfluidic circuit120. Although a single microfluidic circuit 120 is illustrated in FIG.1A, suitable microfluidic devices can include a plurality (e.g., 2 or 3)of such microfluidic circuits. Regardless, the microfluidic device 100can be configured to be a nanofluidic device. As illustrated in FIG. 1A,the microfluidic circuit 120 may include a plurality of microfluidicsequestration chambers 124, 126, 128, and 130, where each sequestrationchamber has one or more openings in fluidic communication with flow path106. In some embodiments of the device of FIG. 1A, the sequestrationchambers have only a single opening in fluidic communication with theflow path 106. As discussed further below, the microfluidicsequestration chambers comprise various features and structures thathave been optimized for retaining micro-objects in the microfluidicdevice, such as microfluidic device 100, even when a medium 180 isflowing through the flow path 106.

Before turning to the foregoing, however, a brief description ofmicrofluidic device 100 and system 150 is provided.

As generally illustrated in FIG. 1A, the microfluidic circuit 120 isdefined by an enclosure 102. Although the enclosure 102 can bephysically structured in different configurations, in the example shownin FIG. 1A the enclosure 102 is depicted as comprising a supportstructure 104 (e.g., a base), a microfluidic circuit structure 108, anda cover 110. The support structure 104, microfluidic circuit structure108, and cover 110 can be attached to each other. For example, themicrofluidic circuit structure 108 can be disposed on an inner surface109 of the support structure 104, and the cover 110 can be disposed overthe microfluidic circuit structure 108. Together with the supportstructure 104 and cover 110, the microfluidic circuit structure 108 candefine the elements of the microfluidic circuit 120.

The support structure 104 can be at the bottom and the cover 110 at thetop of the microfluidic circuit 120 as illustrated in FIG. 1A.Alternatively, the support structure 104 and the cover 110 can beconfigured in other orientations. For example, the support structure 104can be at the top and the cover 110 at the bottom of the microfluidiccircuit 120. Regardless, there can be one or more ports 107 eachcomprising a passage into or out of the enclosure 102. Examples of apassage include a valve, a gate, a pass-through hole, or the like. Asillustrated, port 107 is a pass-through hole created by a gap in themicrofluidic circuit structure 108. However, the port 107 can besituated in other components of the enclosure 102, such as the cover110. Only one port 107 is illustrated in FIG. 1A but the microfluidiccircuit 120 can have two or more ports 107. For example, there can be afirst port 107 that functions as an inlet for fluid entering themicrofluidic circuit 120, and there can be a second port 107 thatfunctions as an outlet for fluid exiting the microfluidic circuit 120.Whether a port 107 function as an inlet or an outlet can depend upon thedirection that fluid flows through flow path 106.

The support structure 104 can comprise one or more electrodes (notshown) and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can comprise one or moresemiconductor substrates, each of which is electrically connected to anelectrode (e.g., all or a subset of the semiconductor substrates can beelectrically connected to a single electrode). The support structure 104can further comprise a printed circuit board assembly (“PCBA”). Forexample, the semiconductor substrate(s) can be mounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can comprise spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with fluid, such as flow regions (which may include or beone or more flow channels), chambers, pens, traps, and the like.

In the microfluidic circuit 120 illustrated in FIG. 1A, the microfluidiccircuit structure 108 comprises a frame 114 and a microfluidic circuitmaterial 116. The frame 114 can partially or completely enclose themicrofluidic circuit material 116. The frame 114 can be, for example, arelatively rigid structure substantially surrounding the microfluidiccircuit material 116. For example, the frame 114 can comprise a metalmaterial.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone or“PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110 can be an integral part of the frame 114 and/or themicrofluidic circuit material 116. Alternatively, the cover 110 can be astructurally distinct element, as illustrated in FIG. 1A. The cover 110can comprise the same or different materials than the frame 114 and/orthe microfluidic circuit material 116. Similarly, the support structure104 can be a separate structure from the frame 114 or microfluidiccircuit material 116 as illustrated, or an integral part of the frame114 or microfluidic circuit material 116. Likewise, the frame 114 andmicrofluidic circuit material 116 can be separate structures as shown inFIG. 1A or integral portions of the same structure.

In some embodiments, the cover 110 can comprise a rigid material. Therigid material may be glass or a material with similar properties. Insome embodiments, the cover 110 can comprise a deformable material. Thedeformable material can be a polymer, such as PDMS. In some embodiments,the cover 110 can comprise both rigid and deformable materials. Forexample, one or more portions of cover 110 (e.g., one or more portionspositioned over sequestration chambers 124, 126, 128, 130) can comprisea deformable material that interfaces with rigid materials of the cover110. In some embodiments, the cover 110 can further include one or moreelectrodes. The one or more electrodes can comprise a conductive oxide,such as indium-tin-oxide (ITO), which may be coated on glass or asimilarly insulating material. Alternatively, the one or more electrodescan be flexible electrodes, such as single-walled nanotubes,multi-walled nanotubes, nanowires, clusters of electrically conductivenanoparticles, or combinations thereof, embedded in a deformablematerial, such as a polymer (e.g., PDMS). Flexible electrodes that canbe used in microfluidic devices have been described, for example, inU.S. 2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light.

The cover 110 may also include at least one material that is gaspermeable (e.g., PDMS or PPS).

FIG. 1A also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150includes an electrical power source 192, an imaging device (incorporatedwithin imaging module 164, where device 194 is not illustrated in FIG.1A, per se), and a tilting device 190 (part of tilting module 166, wheredevice 190 is not illustrated in FIG. 1A).

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, comprise one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device (part ofimaging module 164, discussed below) can comprise a device, such as adigital camera, for capturing images inside microfluidic circuit 120. Insome instances, the imaging device further comprises a detector having afast frame rate and/or high sensitivity (e.g. for low lightapplications). The imaging device can also include a mechanism fordirecting stimulating radiation and/or light beams into the microfluidiccircuit 120 and collecting radiation and/or light beams reflected oremitted from the microfluidic circuit 120 (or micro-objects containedtherein). The emitted light beams may be in the visible spectrum andmay, e.g., include fluorescent emissions. The reflected light beams mayinclude reflected emissions originating from an LED or a wide spectrumlamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or aXenon arc lamp. As discussed with respect to FIG. 3B, the imaging devicemay further include a microscope (or an optical train), which may or maynot include an eyepiece.

System 150 further comprises a tilting device 190 (part of tiltingmodule 166, discussed below) configured to rotate a microfluidic device100 about one or more axes of rotation. In some embodiments, the tiltingdevice 190 is configured to support and/or hold the enclosure 102comprising the microfluidic circuit 120 about at least one axis suchthat the microfluidic device 100 (and thus the microfluidic circuit 120)can be held in a level orientation (i.e. at 0° relative to x- andy-axes), a vertical orientation (i.e. at 90° relative to the x-axisand/or the y-axis), or any orientation therebetween. The orientation ofthe microfluidic device 100 (and the microfluidic circuit 120) relativeto an axis is referred to herein as the “tilt” of the microfluidicdevice 100 (and the microfluidic circuit 120). For example, the tiltingdevice 190 can tilt the microfluidic device 100 at 0.1°, 0.2°, 0.3°,0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°,25°, 30°, 35° 40°, 45° 50°, 55° 60°, 65°, 70°, 75°, 80°, 90° relative tothe x-axis or any degree therebetween. The level orientation (and thusthe x- and y-axes) is defined as normal to a vertical axis defined bythe force of gravity. The tilting device can also tilt the microfluidicdevice 100 (and the microfluidic circuit 120) to any degree greater than90° relative to the x-axis and/or y-axis, or tilt the microfluidicdevice 100 (and the microfluidic circuit 120) 180° relative to thex-axis or the y-axis in order to fully invert the microfluidic device100 (and the microfluidic circuit 120). Similarly, in some embodiments,the tilting device 190 tilts the microfluidic device 100 (and themicrofluidic circuit 120) about an axis of rotation defined by flow path106 or some other portion of microfluidic circuit 120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration chambers. The term “above” as used herein denotesthat the flow path 106 is positioned higher than the one or moresequestration chambers on a vertical axis defined by the force ofgravity (i.e. an object in a sequestration chamber above a flow path 106would have a higher gravitational potential energy than an object in theflow path). The term “below” as used herein denotes that the flow path106 is positioned lower than the one or more sequestration chambers on avertical axis defined by the force of gravity (i.e. an object in asequestration chamber below a flow path 106 would have a lowergravitational potential energy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration chambers without being located directly above or below thesequestration chambers. In other instances, the tilting device 190 tiltsthe microfluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1A.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100.

FIG. 1A also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device (e.g., a camera, microscope, lightsource or any combination thereof) for capturing images (e.g., digitalimages), and a tilting module 166 for controlling a tilting device 190.The control equipment 152 can also include other modules 168 forcontrolling, monitoring, or performing other functions with respect tothe microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can comprise a control module 156 and adigital memory 158. The control module 156 can comprise, for example, adigital processor configured to operate in accordance with machineexecutable instructions (e.g., software, firmware, source code, or thelike) stored as non-transitory data or signals in the memory 158.Alternatively, or in addition, the control module 156 can comprisehardwired digital circuitry and/or analog circuitry. The media module160, motive module 162, imaging module 164, tilting module 166, and/orother modules 168 can be similarly configured. Thus, functions,processes acts, actions, or steps of a process discussed herein as beingperformed with respect to the microfluidic device 100 or any othermicrofluidic apparatus can be performed by any one or more of the mastercontroller 154, media module 160, motive module 162, imaging module 164,tilting module 166, and/or other modules 168 configured as discussedabove. Similarly, the master controller 154, media module 160, motivemodule 162, imaging module 164, tilting module 166, and/or other modules168 may be communicatively coupled to transmit and receive data used inany function, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107). The media module 160 can also control removal of media from theenclosure 102 (e.g., through an outlet port (not shown)). One or moremedia can thus be selectively input into and removed from themicrofluidic circuit 120. The media module 160 can also control the flowof fluidic medium 180 in the flow path 106 inside the microfluidiccircuit 120. For example, in some embodiments media module 160 stops theflow of media 180 in the flow path 106 and through the enclosure 102prior to the tilting module 166 causing the tilting device 190 to tiltthe microfluidic device 100 to a desired angle of incline.

The motive module 162 can be configured to control selection, trapping,and movement of micro-objects (not shown) in the microfluidic circuit120. As discussed below with respect to FIGS. 1B and 1C, the enclosure102 can comprise a dielectrophoresis (DEP), optoelectronic tweezers(OET) and/or opto-electrowetting (OEW) configuration (not shown in FIG.1A), and the motive module 162 can control the activation of electrodesand/or transistors (e.g., phototransistors) to select and movemicro-objects (not shown) and/or droplets of medium (not shown) in theflow path 106 and/or sequestration chambers 124, 126, 128, 130.

The imaging module 164 can control the imaging device. For example, theimaging module 164 can receive and process image data from the imagingdevice. Image data from the imaging device can comprise any type ofinformation captured by the imaging device (e.g., the presence orabsence of micro-objects, droplets of medium, accumulation of label,such as fluorescent label, etc.). Using the information captured by theimaging device, the imaging module 164 can further calculate theposition of objects (e.g., micro-objects, droplets of medium) and/or therate of motion of such objects within the microfluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively, or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration chambers via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1A, the microfluidic circuit 120 isillustrated as comprising a microfluidic channel 122 and sequestrationchambers 124, 126, 128, 130. Each pen comprises an opening to channel122, but otherwise is enclosed such that the pens can substantiallyisolate micro-objects inside the pen from fluidic medium 180 and/ormicro-objects in the flow path 106 of channel 122 or in other pens. Thewalls of the sequestration chamber extend from the inner surface 109 ofthe base to the inside surface of the cover 110 to provide enclosure.The opening of the pen to the microfluidic channel 122 is oriented at anangle to the flow 106 of fluidic medium 180 such that flow 106 is notdirected into the pens. The flow may be tangential or orthogonal to theplane of the opening of the pen. In some instances, pens 124, 126, 128,130 are configured to physically corral one or more micro-objects withinthe microfluidic circuit 120. Sequestration chambers in accordance withthe present disclosure can comprise various shapes, surfaces andfeatures that are optimized for use with DEP, OET, OEW, fluid flow,and/or gravitational forces, as will be discussed and shown in detailbelow.

The microfluidic circuit 120 may comprise any number of microfluidicsequestration chambers. Although five sequestration chambers are shown,microfluidic circuit 120 may have fewer or more sequestration chambers.As used herein, the terms “sequestration chamber” and “sequestrationpen” are used interchangeably. As shown, microfluidic sequestrationchambers 124, 126, 128, and 130 of microfluidic circuit 120 eachcomprise differing features and shapes which may provide one or morebenefits useful in detecting cell-specific antibody secretions, such asisolating one B cell from an adjacent B cell. In some embodiments, themicrofluidic circuit 120 comprises a plurality of identical microfluidicsequestration chambers.

In some embodiments, the microfluidic circuit 120 comprises a pluralityof microfluidic sequestration chambers, wherein two or more of thesequestration chambers comprise differing structures and/or featureswhich provide differing benefits in maintaining different types ofcells. One non-limiting example may include maintaining B cells in onetype of pen while maintaining cancer cells in a different type of pen.

In the embodiment illustrated in FIG. 1A, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to comprise a flow path 106. Themicrofluidic circuit 120 further comprises an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 comprises a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration chambers is configured (e.g., relative to achannel 122) such that the sequestration chambers can be loaded withtarget micro-objects in parallel.

In some embodiments, microfluidic circuit 120 further comprises one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationchambers 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single micro-object from the flowpath 106. In some embodiments, the traps 132 are configured to receiveor capture a plurality of micro-objects from the flow path 106. In someinstances, the traps 132 comprise a volume approximately equal to thevolume of a single target micro-object.

The traps 132 may further comprise an opening which is configured toassist the flow of targeted micro-objects into the traps 132. In someinstances, the traps 132 comprise an opening having a height and widththat is approximately equal to the dimensions of a single targetmicro-object, whereby larger micro-objects are prevented from enteringinto the micro-object trap. The traps 132 may further comprise otherfeatures configured to assist in retention of targeted micro-objectswithin the trap 132. In some instances, the trap 132 is aligned with andsituated on the opposite side of a channel 122 relative to the openingof a microfluidic sequestration chamber, such that upon tilting themicrofluidic device 100 about an axis parallel to the microfluidicchannel 122, the trapped micro-object exits the trap 132 at a trajectorythat causes the micro-object to fall into the opening of thesequestration chamber. In some instances, the trap 132 comprises a sidepassage 134 that is smaller than the target micro-object in order tofacilitate flow through the trap 132 and thereby increase the likelihoodof capturing a micro-object in the trap 132.

In some embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration chambers) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration chamber. In some embodiments, DEP forces are used toprevent a micro-object within a sequestration chamber (e.g.,sequestration pen 124, 126, 128, or 130) from being displaced therefrom.Further, in some embodiments, DEP forces are used to selectively removea micro-object from a sequestration chamber that was previouslycollected in accordance with the embodiments of the current disclosure.In some embodiments, the DEP forces comprise optoelectronic tweezer(OET) forces.

In other embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration chambers) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration chamber. In someembodiments, OEW forces are used to prevent a droplet within asequestration chamber (e.g., sequestration chamber 124, 126, 128, or130) from being displaced therefrom. Further, in some embodiments, OEWforces are used to selectively remove a droplet from a sequestrationchamber that was previously collected in accordance with the embodimentsof the current disclosure.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestrationchamber, and the force of gravity can transport the micro-objects and/ordroplets into the chambers. In some embodiments, the DEP and/or OEWforces can be applied prior to the other forces. In other embodiments,the DEP and/or OEW forces can be applied after the other forces. Instill other instances, the DEP and/or OEW forces can be applied at thesame time as the other forces or in an alternating manner with the otherforces.

FIGS. 1B, 1C, and 2A-2H illustrates various embodiments of microfluidicdevices that can be used in the practice of the embodiments of thepresent disclosure. FIG. 1B depicts an embodiment in which themicrofluidic device 200 is configured as an optically-actuatedelectrokinetic device. A variety of optically-actuated electrokineticdevices are known in the art, including devices having an optoelectronictweezer (OET) configuration and devices having an opto-electrowetting(OEW) configuration. Examples of suitable OET configurations areillustrated in the following U.S. patent documents, each of which isincorporated herein by reference in its entirety: U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355); andU.S. Pat. No. 7,956,339 (Ohta et al.). Examples of OEW configurationsare illustrated in U.S. Pat. No. 6,958,132 (Chiou et al.) and U.S.Patent Application Publication No. 2012/0024708 (Chiou et al.), both ofwhich are incorporated by reference herein in their entirety. Yetanother example of an optically-actuated electrokinetic device includesa combined OET/OEW configuration, examples of which are shown in U.S.Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599(Khandros et al.) and their corresponding PCT Publications WO2015/164846and WO2015/164847, all of which are incorporated herein by reference intheir entirety.

Examples of microfluidic devices having pens in which tumor-derivedcells, such as B cells, T cells, or cancer cells can be placed,cultured, and/or monitored have been described, for example, in US2014/0116881 (application Ser. No. 14/060,117, filed Oct. 22, 2013), US2015/0151298 (application Ser. No. 14/520,568, filed Oct. 22, 2014), andUS 2015/0165436 (application Ser. No. 14/521,447, filed Oct. 22, 2014),each of which is incorporated herein by reference in its entirety. U.S.application Ser. Nos. 14/520,568 and 14/521,447 also describe exemplarymethods of analyzing secretions of cells cultured in a microfluidicdevice. Each of the foregoing applications further describesmicrofluidic devices configured to produce dielectrophoretic (DEP)forces, such as optoelectronic tweezers (OET) or configured to provideopto-electro wetting (OEW). For example, the optoelectronic tweezersdevice illustrated in FIG. 2 of US 2014/0116881 is an example of adevice that can be utilized in embodiments of the present disclosure toselect and move an individual biological micro-object or a group ofbiological micro-objects.

C. Microfluidic Device Motive Configurations.

As described above, the control and monitoring equipment of the systemcan comprise a motive module for selecting and moving objects, such asmicro-objects or droplets, in the microfluidic circuit of a microfluidicdevice. The microfluidic device can have a variety of motiveconfigurations, depending upon the type of object being moved and otherconsiderations. For example, a dielectrophoresis (DEP) configuration canbe utilized to select and move micro-objects in the microfluidiccircuit. Thus, the support structure 104 and/or cover 110 of themicrofluidic device 100 can comprise a DEP configuration for selectivelyinducing DEP forces on micro-objects in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual micro-objects or groups of micro-objects. Alternatively, thesupport structure 104 and/or cover 110 of the microfluidic device 100can comprise an electrowetting (EW) configuration for selectivelyinducing EW forces on droplets in a fluidic medium 180 in themicrofluidic circuit 120 and thereby select, capture, and/or moveindividual droplets or groups of droplets.

One example of a microfluidic device 200 comprising a DEP configurationis illustrated in FIGS. 1B and 1C. While for purposes of simplicityFIGS. 1B and 1C show a side cross-sectional view and a topcross-sectional view, respectively, of a portion of an enclosure 102 ofthe microfluidic device 200 having a region/chamber 202, it should beunderstood that the region/chamber 202 may be part of a fluidic circuitelement having a more detailed structure, such as a growth chamber, asequestration pen, a flow region, or a flow channel. Furthermore, themicrofluidic device 200 may include other fluidic circuit elements. Forexample, the microfluidic device 200 can include a plurality of growthchambers or sequestration pens and/or one or more flow regions or flowchannels, such as those described herein with respect to microfluidicdevice 100. A DEP configuration may be incorporated into any suchfluidic circuit elements of the microfluidic device 200, or selectportions thereof. It should be further appreciated that any of the aboveor below described microfluidic device components and system componentsmay be incorporated in and/or used in combination with the microfluidicdevice 200. For example, system 150 including control and monitoringequipment 152, described above, may be used with microfluidic device200, including one or more of the media module 160, motive module 162,imaging module 164, tilting module 166, and other modules 168.

As seen in FIG. 1B, the microfluidic device 200 includes a supportstructure 104 having a bottom electrode 204 and an electrode activationsubstrate 206 overlying the bottom electrode 204, and a cover 110 havinga top electrode 210, with the top electrode 210 spaced apart from thebottom electrode 204. The top electrode 210 and the electrode activationsubstrate 206 define opposing surfaces of the region/chamber 202. Amedium 180 contained in the region/chamber 202 thus provides a resistiveconnection between the top electrode 210 and the electrode activationsubstrate 206. A power source 212 configured to be connected to thebottom electrode 204 and the top electrode 210 and create a biasingvoltage between the electrodes, as required for the generation of DEPforces in the region/chamber 202, is also shown. The power source 212can be, for example, an alternating current (AC) power source.

In certain embodiments, the microfluidic device 200 illustrated in FIGS.1B and 1C can have an optically-actuated DEP configuration. Accordingly,changing patterns of light 218 from the light source 216, which may becontrolled by the motive module 162, can selectively activate anddeactivate changing patterns of DEP electrodes at regions 214 of theinner surface 208 of the electrode activation substrate 206.(Hereinafter the regions 214 of a microfluidic device having a DEPconfiguration are referred to as “DEP electrode regions.”) Asillustrated in FIG. 1C, a light pattern 218 directed onto the innersurface 208 of the electrode activation substrate 206 can illuminateselect DEP electrode regions 214 a (shown in white) in a pattern, suchas a square. The non-illuminated DEP electrode regions 214(cross-hatched) are hereinafter referred to as “dark” DEP electroderegions 214. The relative electrical impedance through the DEP electrodeactivation substrate 206 (i.e., from the bottom electrode 204 up to theinner surface 208 of the electrode activation substrate 206 whichinterfaces with the medium 180 in the flow region 106) is greater thanthe relative electrical impedance through the medium 180 in theregion/chamber 202 (i.e., from the inner surface 208 of the electrodeactivation substrate 206 to the top electrode 210 of the cover 110) ateach dark DEP electrode region 214. An illuminated DEP electrode region214 a, however, exhibits a reduced relative impedance through theelectrode activation substrate 206 that is less than the relativeimpedance through the medium 180 in the region/chamber 202 at eachilluminated DEP electrode region 214 a.

With the power source 212 activated, the foregoing DEP configurationcreates an electric field gradient in the fluidic medium 180 betweenilluminated DEP electrode regions 214 a and adjacent dark DEP electroderegions 214, which in turn creates local DEP forces that attract orrepel nearby micro-objects (not shown) in the fluidic medium 180. DEPelectrodes that attract or repel micro-objects in the fluidic medium 180can thus be selectively activated and deactivated at many different suchDEP electrode regions 214 at the inner surface 208 of the region/chamber202 by changing light patterns 218 projected from a light source 216into the microfluidic device 200. Whether the DEP forces attract orrepel nearby micro-objects can depend on such parameters as thefrequency of the power source 212 and the dielectric properties of themedium 180 and/or micro-objects (not shown).

The square pattern 220 of illuminated DEP electrode regions 214 aillustrated in FIG. 1C is an example only. Any pattern of the DEPelectrode regions 214 can be illuminated (and thereby activated) by thepattern of light 218 projected into the microfluidic device 200, and thepattern of illuminated/activated DEP electrode regions 214 can berepeatedly changed by changing or moving the light pattern 218.

In some embodiments, the electrode activation substrate 206 can compriseor consist of a photoconductive material. In such embodiments, the innersurface 208 of the electrode activation substrate 206 can befeatureless. For example, the electrode activation substrate 206 cancomprise or consist of a layer of hydrogenated amorphous silicon(a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen(calculated as 100*the number of hydrogen atoms/the total number ofhydrogen and silicon atoms). The layer of a-Si:H can have a thickness ofabout 500 nm to about 2.0 μm. In such embodiments, the DEP electroderegions 214 can be created anywhere and in any pattern on the innersurface 208 of the electrode activation substrate 206, in accordancewith the light pattern 218. The number and pattern of the DEP electroderegions 214 thus need not be fixed, but can correspond to the lightpattern 218. Examples of microfluidic devices having a DEP configurationcomprising a photoconductive layer such as discussed above have beendescribed, for example, in U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355), the entire contents ofwhich are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 cancomprise a substrate comprising a plurality of doped layers,electrically insulating layers (or regions), and electrically conductivelayers that form semiconductor integrated circuits, such as is known insemiconductor fields. For example, the electrode activation substrate206 can comprise a plurality of phototransistors, including, forexample, lateral bipolar phototransistors, each phototransistorcorresponding to a DEP electrode region 214. Alternatively, theelectrode activation substrate 206 can comprise electrodes (e.g.,conductive metal electrodes) controlled by phototransistor switches,with each such electrode corresponding to a DEP electrode region 214.The electrode activation substrate 206 can include a pattern of suchphototransistors or phototransistor-controlled electrodes. The pattern,for example, can be an array of substantially square phototransistors orphototransistor-controlled electrodes arranged in rows and columns, suchas shown in FIG. 2B. Alternatively, the pattern can be an array ofsubstantially hexagonal phototransistors or phototransistor-controlledelectrodes that form a hexagonal lattice. Regardless of the pattern,electric circuit elements can form electrical connections between theDEP electrode regions 214 at the inner surface 208 of the electrodeactivation substrate 206 and the bottom electrode 210, and thoseelectrical connections (i.e., phototransistors or electrodes) can beselectively activated and deactivated by the light pattern 218. When notactivated, each electrical connection can have high impedance such thatthe relative impedance through the electrode activation substrate 206(i.e., from the bottom electrode 204 to the inner surface 208 of theelectrode activation substrate 206 which interfaces with the medium 180in the region/chamber 202) is greater than the relative impedancethrough the medium 180 (i.e., from the inner surface 208 of theelectrode activation substrate 206 to the top electrode 210 of the cover110) at the corresponding DEP electrode region 214. When activated bylight in the light pattern 218, however, the relative impedance throughthe electrode activation substrate 206 is less than the relativeimpedance through the medium 180 at each illuminated DEP electroderegion 214, thereby activating the DEP electrode at the correspondingDEP electrode region 214 as discussed above. DEP electrodes that attractor repel micro-objects (not shown) in the medium 180 can thus beselectively activated and deactivated at many different DEP electroderegions 214 at the inner surface 208 of the electrode activationsubstrate 206 in the region/chamber 202 in a manner determined by thelight pattern 218.

Examples of microfluidic devices having electrode activation substratesthat comprise phototransistors have been described, for example, in U.S.Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device 300 illustrated inFIGS. 21 and 22, and descriptions thereof), and U.S. Patent PublicationNo. 2016/0184821 (Hobbs et al.) (see, e.g., devices 200, 502, 504, 600,and 700 illustrated throughout the drawings, and descriptions thereof),the entire contents of each of which are incorporated herein byreference. Examples of microfluidic devices having electrode activationsubstrates that comprise electrodes controlled by phototransistorswitches have been described, for example, in U.S. Patent PublicationNo. 2014/0124370 (Short et al.) (see, e.g., devices 200, 400, 500, 600,and 900 illustrated throughout the drawings, and descriptions thereof),the entire contents of which are incorporated herein by reference.

In some embodiments of a DEP configured microfluidic device, the topelectrode 210 is part of a first wall (or cover 110) of the enclosure102, and the electrode activation substrate 206 and bottom electrode 204are part of a second wall (or support structure 104) of the enclosure102. The region/chamber 202 can be between the first wall and the secondwall. In other embodiments, the electrode 210 is part of the second wall(or support structure 104) and one or both of the electrode activationsubstrate 206 and/or the electrode 210 are part of the first wall (orcover 110). Moreover, the light source 216 can alternatively be used toilluminate the enclosure 102 from below.

With the microfluidic device 200 of FIGS. 1B-1C having a DEPconfiguration, the motive module 162 can select a micro-object (notshown) in the medium 180 in the region/chamber 202 by projecting a lightpattern 218 into the microfluidic device 200 to activate a first set ofone or more DEP electrodes at DEP electrode regions 214 a of the innersurface 208 of the electrode activation substrate 206 in a pattern(e.g., square pattern 220) that surrounds and captures the micro-object.The motive module 162 can then move the in situ-generated capturedmicro-object by moving the light pattern 218 relative to themicrofluidic device 200 to activate a second set of one or more DEPelectrodes at DEP electrode regions 214. Alternatively, the microfluidicdevice 200 can be moved relative to the light pattern 218.

In other embodiments, the microfluidic device 200 can have a DEPconfiguration that does not rely upon light activation of DEP electrodesat the inner surface 208 of the electrode activation substrate 206. Forexample, the electrode activation substrate 206 can comprise selectivelyaddressable and energizable electrodes positioned opposite to a surfaceincluding at least one electrode (e.g., cover 110). Switches (e.g.,transistor switches in a semiconductor substrate) may be selectivelyopened and closed to activate or inactivate DEP electrodes at DEPelectrode regions 214, thereby creating a net DEP force on amicro-object (not shown) in region/chamber 202 in the vicinity of theactivated DEP electrodes. Depending on such characteristics as thefrequency of the power source 212 and the dielectric properties of themedium (not shown) and/or micro-objects in the region/chamber 202, theDEP force can attract or repel a nearby micro-object. By selectivelyactivating and deactivating a set of DEP electrodes (e.g., at a set ofDEP electrodes regions 214 that forms a square pattern 220), one or moremicro-objects in region/chamber 202 can be trapped and moved within theregion/chamber 202. The motive module 162 in FIG. 1A can control suchswitches and thus activate and deactivate individual ones of the DEPelectrodes to select, trap, and move particular micro-objects (notshown) around the region/chamber 202. Microfluidic devices having a DEPconfiguration that includes selectively addressable and energizableelectrodes are known in the art and have been described, for example, inU.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776(Medoro), the entire contents of which are incorporated herein byreference.

As yet another example, the microfluidic device 200 can have anelectrowetting (EW) configuration, which can be in place of the DEPconfiguration or can be located in a portion of the microfluidic device200 that is separate from the portion which has the DEP configuration.The EW configuration can be an opto-electrowetting configuration or anelectrowetting on dielectric (EWOD) configuration, both of which areknown in the art. In some EW configurations, the support structure 104has an electrode activation substrate 206 sandwiched between adielectric layer (not shown) and the bottom electrode 204. Thedielectric layer can comprise a hydrophobic material and/or can becoated with a hydrophobic material, as described below. For microfluidicdevices 200 that have an EW configuration, the inner surface 208 of thesupport structure 104 is the inner surface of the dielectric layer orits hydrophobic coating.

The dielectric layer (not shown) can comprise one or more oxide layers,and can have a thickness of about 50 nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer maycomprise a layer of oxide, such as a metal oxide (e.g., aluminum oxideor hafnium oxide). In certain embodiments, the dielectric layer cancomprise a dielectric material other than a metal oxide, such as siliconoxide or a nitride. Regardless of the exact composition and thickness,the dielectric layer can have an impedance of about 10 kOhms to about 50kOhms.

In some embodiments, the surface of the dielectric layer that facesinward toward region/chamber 202 is coated with a hydrophobic material.The hydrophobic material can comprise, for example, fluorinated carbonmolecules. Examples of fluorinated carbon molecules includeperfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™.Molecules that make up the hydrophobic material can be covalently bondedto the surface of the dielectric layer. For example, molecules of thehydrophobic material can be covalently bound to the surface of thedielectric layer by means of a linker such as a siloxane group, aphosphonic acid group, or a thiol group. Thus, in some embodiments, thehydrophobic material can comprise alkyl-terminated siloxane,alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkylgroup can be long-chain hydrocarbons (e.g., having a chain of at least10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively,fluorinated (or perfluorinated) carbon chains can be used in place ofthe alkyl groups. Thus, for example, the hydrophobic material cancomprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminatedphosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments,the hydrophobic coating has a thickness of about 10 nm to about 50 nm.In other embodiments, the hydrophobic coating has a thickness of lessthan 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm).

In some embodiments, the cover 110 of a microfluidic device 200 havingan electrowetting configuration is coated with a hydrophobic material(not shown) as well. The hydrophobic material can be the samehydrophobic material used to coat the dielectric layer of the supportstructure 104, and the hydrophobic coating can have a thickness that issubstantially the same as the thickness of the hydrophobic coating onthe dielectric layer of the support structure 104. Moreover, the cover110 can comprise an electrode activation substrate 206 sandwichedbetween a dielectric layer and the top electrode 210, in the manner ofthe support structure 104. The electrode activation substrate 206 andthe dielectric layer of the cover 110 can have the same compositionand/or dimensions as the electrode activation substrate 206 and thedielectric layer of the support structure 104. Thus, the microfluidicdevice 200 can have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 can comprisea photoconductive material, such as described above. Accordingly, incertain embodiments, the electrode activation substrate 206 can compriseor consist of a layer of hydrogenated amorphous silicon (a-Si:H). Thea-Si:H can comprise, for example, about 8% to 40% hydrogen (calculatedas 100*the number of hydrogen atoms/the total number of hydrogen andsilicon atoms). The layer of a-Si:H can have a thickness of about 500 nmto about 2.0 μm. Alternatively, the electrode activation substrate 206can comprise electrodes (e.g., conductive metal electrodes) controlledby phototransistor switches, as described above. Microfluidic deviceshaving an opto-electrowetting configuration are known in the art and/orcan be constructed with electrode activation substrates known in theart. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entirecontents of which are incorporated herein by reference, disclosesopto-electrowetting configurations having a photoconductive materialsuch as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short etal.), referenced above, discloses electrode activation substrates havingelectrodes controlled by phototransistor switches.

The microfluidic device 200 thus can have an opto-electrowettingconfiguration, and light patterns 218 can be used to activatephotoconductive EW regions or photoresponsive EW electrodes in theelectrode activation substrate 206. Such activated EW regions or EWelectrodes of the electrode activation substrate 206 can generate anelectrowetting force at the inner surface 208 of the support structure104 (i.e., the inner surface of the overlaying dielectric layer or itshydrophobic coating). By changing the light patterns 218 (or movingmicrofluidic device 200 relative to the light source 216) incident onthe electrode activation substrate 206, droplets (e.g., containing anaqueous medium, solution, or solvent) contacting the inner surface 208of the support structure 104 can be moved through an immiscible fluid(e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic devices 200 can have an EWODconfiguration, and the electrode activation substrate 206 can compriseselectively addressable and energizable electrodes that do not rely uponlight for activation. The electrode activation substrate 206 thus caninclude a pattern of such electrowetting (EW) electrodes. The pattern,for example, can be an array of substantially square EW electrodesarranged in rows and columns, such as shown in FIG. 2B. Alternatively,the pattern can be an array of substantially hexagonal EW electrodesthat form a hexagonal lattice. Regardless of the pattern, the EWelectrodes can be selectively activated (or deactivated) by electricalswitches (e.g., transistor switches in a semiconductor substrate). Byselectively activating and deactivating EW electrodes in the electrodeactivation substrate 206, droplets (not shown) contacting the innersurface 208 of the overlaying dielectric layer or its hydrophobiccoating can be moved within the region/chamber 202. The motive module162 in FIG. 1A can control such switches and thus activate anddeactivate individual EW electrodes to select and move particulardroplets around region/chamber 202. Microfluidic devices having a EWODconfiguration with selectively addressable and energizable electrodesare known in the art and have been described, for example, in U.S. Pat.No. 8,685,344 (Sundarsan et al.), the entire contents of which areincorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, a powersource 212 can be used to provide a potential (e.g., an AC voltagepotential) that powers the electrical circuits of the microfluidicdevice 200. The power source 212 can be the same as, or a component of,the power source 192 referenced in FIG. 1. Power source 212 can beconfigured to provide an AC voltage and/or current to the top electrode210 and the bottom electrode 204. For an AC voltage, the power source212 can provide a frequency range and an average or peak power (e.g.,voltage or current) range sufficient to generate net DEP forces (orelectrowetting forces) strong enough to trap and move individualmicro-objects (not shown) in the region/chamber 202, as discussed above,and/or to change the wetting properties of the inner surface 208 of thesupport structure 104 (i.e., the dielectric layer and/or the hydrophobiccoating on the dielectric layer) in the region/chamber 202, as alsodiscussed above. Such frequency ranges and average or peak power rangesare known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.),U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No.7,612,355), and US Patent Application Publication Nos. US2014/0124370(Short et al.), US2015/0306598 (Khandros et al.), and US2015/0306599(Khandros et al.).

D. Sequestration Chambers.

Non-limiting examples of generic sequestration chamber 224, 226, and 228are shown within the microfluidic device 230 depicted in FIGS. 2A-2C.Each sequestration chamber 224, 226, and 228 can comprise an isolationstructure 232 defining an isolation region 240 and a connection region236 fluidically connecting the isolation region 240 to a channel 122.The connection region 236 can comprise a proximal opening 234 to themicrofluidic channel 122 and a distal opening 238 to the isolationregion 240. The connection region 236 can be configured so that themaximum penetration depth of a flow of a fluidic medium (not shown)flowing from the microfluidic channel 122 into the sequestration chamber224, 226, 228 does not extend into the isolation region 240. Thus, dueto the connection region 236, a micro-object (not shown) or othermaterial (not shown) disposed in an isolation region 240 of asequestration chamber 224, 226, 228 can thus be isolated from, and notsubstantially affected by, a flow of medium 180 in the microfluidicchannel 122.

The sequestration chambers 224, 226, and 228 of FIGS. 2A-2C each have asingle opening which opens directly to the microfluidic channel 122. Theopening of the sequestration chamber opens laterally from themicrofluidic channel 122. The electrode activation substrate 206underlays both the microfluidic channel 122 and the sequestrationchambers 224, 226, and 228. The upper surface of the electrodeactivation substrate 206 within the enclosure of a sequestrationchamber, forming the floor of the sequestration chamber, is disposed atthe same level or substantially the same level of the upper surface theof electrode activation substrate 206 within the microfluidic channel122 (or flow region if a channel is not present), forming the floor ofthe flow channel (or flow region, respectively) of the microfluidicdevice. The electrode activation substrate 206 may be featureless or mayhave an irregular or patterned surface that varies from its highestelevation to its lowest depression by less than about 3 microns, 2.5microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4microns, 0.2 microns, 0.1 microns or less. The variation of elevation inthe upper surface of the substrate across both the microfluidic channel122 (or flow region) and sequestration chambers may be less than about3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls ofthe sequestration chamber or walls of the microfluidic device. Whiledescribed in detail for the microfluidic device 200, this also appliesto any of the microfluidic devices 100, 230, 250, 280, 290, 320, 400,450, 500, 700 described herein.

The microfluidic channel 122 can thus be an example of a swept region,and the isolation regions 240 of the sequestration chambers 224, 226,228 can be examples of unswept regions. As noted, the microfluidicchannel 122 and sequestration chambers 224, 226, 228 can be configuredto contain one or more fluidic media 180. In the example shown in FIGS.2A-2B, the ports 222 are connected to the microfluidic channel 122 andallow a fluidic medium 180 to be introduced into or removed from themicrofluidic device 230. Prior to introduction of the fluidic medium180, the microfluidic device may be primed with a gas such as carbondioxide gas. Once the microfluidic device 230 contains the fluidicmedium 180, the flow 242 of fluidic medium 180 in the microfluidicchannel 122 can be selectively generated and stopped. For example, asshown, the ports 222 can be disposed at different locations (e.g.,opposite ends) of the microfluidic channel 122, and a flow 242 of mediumcan be created from one port 222 functioning as an inlet to another port222 functioning as an outlet.

FIG. 2C illustrates a detailed view of an example of a sequestrationchamber 224 according to the present disclosure. Examples ofmicro-objects 246 are also shown.

As is known, a flow 242 of fluidic medium 180 in a microfluidic channel122 past a proximal opening 234 of sequestration chamber 224 can cause asecondary flow 244 of the medium 180 into and/or out of thesequestration chamber 224. To isolate micro-objects 246 in the isolationregion 240 of a sequestration chamber 224 from the secondary flow 244,the length L_(con) of the connection region 236 of the sequestrationchamber 224 (i.e., from the proximal opening 234 to the distal opening238) should be greater than the penetration depth D_(p) of the secondaryflow 244 into the connection region 236. The penetration depth D_(p) ofthe secondary flow 244 depends upon the velocity of the fluidic medium180 flowing in the microfluidic channel 122 and various parametersrelating to the configuration of the microfluidic channel 122 and theproximal opening 234 of the connection region 236 to the microfluidicchannel 122. For a given microfluidic device, the configurations of themicrofluidic channel 122 and the opening 234 will be fixed, whereas therate of flow 242 of fluidic medium 180 in the microfluidic channel 122will be variable. Accordingly, for each sequestration chamber 224, amaximal velocity V_(max) for the flow 242 of fluidic medium 180 inchannel 122 can be identified that ensures that the penetration depthD_(p) of the secondary flow 244 does not exceed the length L_(con) ofthe connection region 236. As long as the rate of the flow 242 offluidic medium 180 in the microfluidic channel 122 does not exceed themaximum velocity V_(max), the resulting secondary flow 244 can belimited to the microfluidic channel 122 and the connection region 236and kept out of the isolation region 240. The flow 242 of medium 180 inthe microfluidic channel 122 will thus not draw micro-objects 246 out ofthe isolation region 240. Rather, micro-objects 246 located in theisolation region 240 will stay in the isolation region 240 regardless ofthe flow 242 of fluidic medium 180 in the microfluidic channel 122.

Moreover, as long as the rate of flow 242 of medium 180 in themicrofluidic channel 122 does not exceed V_(max), the flow 242 offluidic medium 180 in the microfluidic channel 122 will not movemiscellaneous particles (e.g., microparticles and/or nanoparticles) fromthe microfluidic channel 122 into the isolation region 240 of asequestration chamber 224. Having the length L_(con) of the connectionregion 236 be greater than the maximum penetration depth D_(p) of thesecondary flow 244 can thus prevent contamination of one sequestrationchamber 224 with miscellaneous particles from the microfluidic channel122 or another sequestration chamber (e.g., sequestration chambers 226,228 in FIG. 2D).

Because the microfluidic channel 122 and the connection regions 236 ofthe sequestration chambers 224, 226, 228 can be affected by the flow 242of medium 180 in the microfluidic channel 122, the microfluidic channel122 and connection regions 236 can be deemed swept (or flow) regions ofthe microfluidic device 230. The isolation regions 240 of thesequestration chambers 224, 226, 228, on the other hand, can be deemedunswept (or non-flow) regions. For example, components (not shown) in afirst fluidic medium 180 in the microfluidic channel 122 can mix with asecond fluidic medium 248 in the isolation region 240 substantially onlyby diffusion of components of the first medium 180 from the microfluidicchannel 122 through the connection region 236 and into the secondfluidic medium 248 in the isolation region 240. Similarly, components(not shown) of the second medium 248 in the isolation region 240 can mixwith the first medium 180 in the microfluidic channel 122 substantiallyonly by diffusion of components of the second medium 248 from theisolation region 240 through the connection region 236 and into thefirst medium 180 in the microfluidic channel 122. In some embodiments,the extent of fluidic medium exchange between the isolation region of asequestration chamber and the flow region by diffusion is greater thanabout 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about99% of fluidic exchange. The first medium 180 can be the same medium ora different medium than the second medium 248. Moreover, the firstmedium 180 and the second medium 248 can start out being the same, thenbecome different (e.g., through conditioning of the second medium 248 byone or more cells in the isolation region 240, or by changing the medium180 flowing through the microfluidic channel 122).

The maximum penetration depth D_(p) of the secondary flow 244 caused bythe flow 242 of fluidic medium 180 in the microfluidic channel 122 candepend on a number of parameters, as mentioned above. Examples of suchparameters include: the shape of the microfluidic channel 122 (e.g., themicrofluidic channel can direct medium into the connection region 236,divert medium away from the connection region 236, or direct medium in adirection substantially perpendicular to the proximal opening 234 of theconnection region 236 to the microfluidic channel 122); a width W_(ch)(or cross-sectional area) of the microfluidic channel 122 at theproximal opening 234; and a width W_(con) (or cross-sectional area) ofthe connection region 236 at the proximal opening 234; the velocity V ofthe flow 242 of fluidic medium 180 in the microfluidic channel 122; theviscosity of the first medium 180 and/or the second medium 248, or thelike.

In some embodiments, the dimensions of the microfluidic channel 122 andsequestration chambers 224, 226, 228 can be oriented as follows withrespect to the vector of the flow 242 of fluidic medium 180 in themicrofluidic channel 122: the microfluidic channel width W_(ch) (orcross-sectional area of the microfluidic channel 122) can besubstantially perpendicular to the flow 242 of medium 180; the widthW_(con) (or cross-sectional area) of the connection region 236 atopening 234 can be substantially parallel to the flow 242 of medium 180in the microfluidic channel 122; and/or the length L_(con) of theconnection region can be substantially perpendicular to the flow 242 ofmedium 180 in the microfluidic channel 122. The foregoing are examplesonly, and the relative position of the microfluidic channel 122 andsequestration chambers 224, 226, 228 can be in other orientations withrespect to each other.

As illustrated in FIG. 2C, the width W_(con) of the connection region236 can be uniform from the proximal opening 234 to the distal opening238. The width W_(con) of the connection region 236 at the distalopening 238 can thus be in any of the ranges identified herein for thewidth W_(con) of the connection region 236 at the proximal opening 234.Alternatively, the width W_(con) of the connection region 236 at thedistal opening 238 can be larger than the width W_(con) of theconnection region 236 at the proximal opening 234.

As illustrated in FIG. 2C, the width of the isolation region 240 at thedistal opening 238 can be substantially the same as the width W_(con) ofthe connection region 236 at the proximal opening 234. The width of theisolation region 240 at the distal opening 238 can thus be in any of theranges identified herein for the width W_(con) of the connection region236 at the proximal opening 234. Alternatively, the width of theisolation region 240 at the distal opening 238 can be larger or smallerthan the width W_(con) of the connection region 236 at the proximalopening 234. Moreover, the distal opening 238 may be smaller than theproximal opening 234 and the width W_(con) of the connection region 236may be narrowed between the proximal opening 234 and distal opening 238.For example, the connection region 236 may be narrowed between theproximal opening and the distal opening, using a variety of differentgeometries (e.g. chamfering the connection region, beveling theconnection region). Further, any part or subpart of the connectionregion 236 may be narrowed (e.g. a portion of the connection regionadjacent to the proximal opening 234).

FIGS. 2D-2F depict another exemplary embodiment of a microfluidic device250 containing a microfluidic circuit 262 and flow channels 264, whichare variations of the respective microfluidic device 100, circuit 132and channel 134 of FIG. 1A. The microfluidic device 250 also has aplurality of sequestration chambers 266 that are additional variationsof the above-described sequestration chambers 124, 126, 128, 130, 224,226 or 228. In particular, it should be appreciated that thesequestration chambers 266 of device 250 shown in FIGS. 2D-2F canreplace any of the above-described sequestration chambers 124, 126, 128,130, 224, 226 or 228 in devices 100, 200, 230, 280, 290, 300. Likewise,the microfluidic device 250 is another variant of the microfluidicdevice 100, and may also have the same or a different DEP configurationas the above-described microfluidic device 100, 200, 230, 280, 290, 300,as well as any of the other microfluidic system components describedherein.

The microfluidic device 250 of FIGS. 2D-2F comprises a support structure(not visible in FIGS. 2D-2F, but can be the same or generally similar tothe support structure 104 of device 100 depicted in FIG. 1A), amicrofluidic circuit structure 256, and a cover (not visible in FIGS.2D-2F, but can be the same or generally similar to the cover 122 ofdevice 100 depicted in FIG. 1A). The microfluidic circuit structure 256includes a frame 252 and microfluidic circuit material 260, which can bethe same as or generally similar to the frame 114 and microfluidiccircuit material 116 of device 100 shown in FIG. 1A. As shown in FIG.2D, the microfluidic circuit 262 defined by the microfluidic circuitmaterial 260 can comprise multiple channels 264 (two are shown but therecan be more) to which multiple sequestration chambers 266 arefluidically connected.

Each sequestration chamber 266 can comprise an isolation structure 272,an isolation region 270 within the isolation structure 272, and aconnection region 268. From a proximal opening 274 at the microfluidicchannel 264 to a distal opening 276 at the isolation structure 272, theconnection region 268 fluidically connects the microfluidic channel 264to the isolation region 270. Generally, in accordance with the abovediscussion of FIGS. 2B and 2C, a flow 278 of a first fluidic medium 254in a channel 264 can create secondary flows 282 of the first medium 254from the microfluidic channel 264 into and/or out of the respectiveconnection regions 268 of the sequestration chambers 266.

As illustrated in FIG. 2E, the connection region 268 of eachsequestration chamber 266 generally includes the area extending betweenthe proximal opening 274 to a channel 264 and the distal opening 276 toan isolation structure 272. The length L_(con) of the connection region268 can be greater than the maximum penetration depth D_(p) of secondaryflow 282, in which case the secondary flow 282 will extend into theconnection region 268 without being redirected toward the isolationregion 270 (as shown in FIG. 2D). Alternatively, at illustrated in FIG.2F, the connection region 268 can have a length L_(con) that is lessthan the maximum penetration depth D_(p), in which case the secondaryflow 282 will extend through the connection region 268 and be redirectedtoward the isolation region 270. In this latter situation, the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than themaximum penetration depth D_(p), so that secondary flow 282 will notextend into isolation region 270. Whether length L_(con) of connectionregion 268 is greater than the penetration depth D_(p), or the sum oflengths L_(c1) and L_(c2) of connection region 268 is greater than thepenetration depth D_(p), a flow 278 of a first medium 254 in channel 264that does not exceed a maximum velocity V_(max) will produce a secondaryflow having a penetration depth D_(p), and micro-objects (not shown butcan be the same or generally similar to the micro-objects 246 shown inFIG. 2C) in the isolation region 270 of a sequestration chamber 266 willnot be drawn out of the isolation region 270 by a flow 278 of firstmedium 254 in channel 264. Nor will the flow 278 in channel 264 drawmiscellaneous materials (not shown) from channel 264 into the isolationregion 270 of a sequestration chamber 266. As such, diffusion is theonly mechanism by which components in a first medium 254 in themicrofluidic channel 264 can move from the microfluidic channel 264 intoa second medium 258 in an isolation region 270 of a sequestrationchamber 266. Likewise, diffusion is the only mechanism by whichcomponents in a second medium 258 in an isolation region 270 of asequestration chamber 266 can move from the isolation region 270 to afirst medium 254 in the microfluidic channel 264. The first medium 254can be the same medium as the second medium 258, or the first medium 254can be a different medium than the second medium 258. Alternatively, thefirst medium 254 and the second medium 258 can start out being the same,then become different, e.g., through conditioning of the second mediumby one or more cells in the isolation region 270, or by changing themedium flowing through the microfluidic channel 264.

As illustrated in FIG. 2E, the width W_(ch) of the microfluidic channels264 (i.e., taken transverse to the direction of a fluid medium flowthrough the microfluidic channel indicated by arrows 278 in FIG. 2D) inthe microfluidic channel 264 can be substantially perpendicular to awidth W_(con1) of the proximal opening 274 and thus substantiallyparallel to a width W_(con2) of the distal opening 276. The widthW_(con1) of the proximal opening 274 and the width W_(con2) of thedistal opening 276, however, need not be substantially perpendicular toeach other. For example, an angle between an axis (not shown) on whichthe width W_(con1) of the proximal opening 274 is oriented and anotheraxis on which the width W_(con2) of the distal opening 276 is orientedcan be other than perpendicular and thus other than 90°. Examples ofalternatively oriented angles include angles in any of the followingranges: from about 300 to about 90°, from about 45 to about 90°, fromabout 600 to about 90°, or the like.

In various embodiments of sequestration chambers (e.g. 124, 126, 128,130, 224, 226, 228, or 266), the isolation region (e.g. 240 or 270) isconfigured to contain a plurality of micro-objects. In otherembodiments, the isolation region can be configured to contain only one,two, three, four, five, or a similar relatively small number ofmicro-objects. Accordingly, the volume of an isolation region can be,for example, at least 1×10⁶, 2×10⁶, 4×10⁶, 6×10⁶ cubic microns, or more.

In various embodiments of sequestration chambers, the width W_(ch) ofthe microfluidic channel (e.g., 122) at a proximal opening (e.g. 234)can be within any of the following ranges: about 50-1000 microns, 50-500microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns,50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns,90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300microns, 100-250 microns, 100-200 microns, 100-150 microns, and 100-120microns. In some other embodiments, the width W_(ch) of the microfluidicchannel (e.g., 122) at a proximal opening (e.g. 234) can be in a rangeof about 200-800 microns, 200-700 microns, or 200-600 microns. Theforegoing are examples only, and the width W_(ch) of the microfluidicchannel 122 can be in other ranges (e.g., a range defined by any of theendpoints listed above). Moreover, the W_(ch) of the microfluidicchannel 122 can be selected to be in any of these ranges in regions ofthe microfluidic channel other than at a proximal opening of asequestration chamber.

In some embodiments, a sequestration chamber has a height of about 30 toabout 200 microns, or about 50 to about 150 microns. In someembodiments, the sequestration chamber has a cross-sectional area ofabout 1×10⁴-3×10⁶ square microns, 2×10⁴-2×10⁶ square microns,4×10⁴-1×10⁶ square microns, 2×10⁴-5×10⁵ square microns, 2×10⁴-1×10⁵square microns or about 2×10⁵-2×10⁶ square microns.

In various embodiments of sequestration chambers, the height H_(ch) ofthe microfluidic channel (e.g., 122) at a proximal opening (e.g., 234)can be within any of the following ranges: 20-100 microns, 20-90microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns,30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns,40-70 microns, 40-60 microns, or 40-50 microns. The foregoing areexamples only, and the height H_(ch) of the microfluidic channel (e.g.,122) can be in other ranges (e.g., a range defined by any of theendpoints listed above). The height H_(ch) of the microfluidic channel122 can be selected to be in any of these ranges in regions of themicrofluidic channel other than at a proximal opening of ansequestration chamber.

In various embodiments of sequestration chamber a cross-sectional areaof the microfluidic channel (e.g., 122) at a proximal opening (e.g.,234) can be within any of the following ranges: 500-50,000 squaremicrons, 500-40,000 square microns, 500-30,000 square microns,500-25,000 square microns, 500-20,000 square microns, 500-15,000 squaremicrons, 500-10,000 square microns, 500-7,500 square microns, 500-5,000square microns, 1,000-25,000 square microns, 1,000-20,000 squaremicrons, 1,000-15,000 square microns, 1,000-10,000 square microns,1,000-7,500 square microns, 1,000-5,000 square microns, 2,000-20,000square microns, 2,000-15,000 square microns, 2,000-10,000 squaremicrons, 2,000-7,500 square microns, 2,000-6,000 square microns,3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000square microns, 3,000-7,500 square microns, or 3,000 to 6,000 squaremicrons. The foregoing are examples only, and the cross-sectional areaof the microfluidic channel (e.g., 122) at a proximal opening (e.g.,234) can be in other ranges (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration chambers, the length L_(con) ofthe connection region (e.g., 236) can be in any of the following ranges:about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns,20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200microns, or about 100-150 microns. The foregoing are examples only, andlength L_(con) of a connection region (e.g., 236) can be in a differentrange than the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration chambers the width W_(con) of aconnection region (e.g., 236) at a proximal opening (e.g., 234) can bein any of the following ranges: 20-500 microns, 20-400 microns, 20-300microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns,20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns,40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns,50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80microns, 70-150 microns, 70-100 microns, and 80-100 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion (e.g., 236) at a proximal opening (e.g., 234) can be differentthan the foregoing examples (e.g., a range defined by any of theendpoints listed above).

In various embodiments of sequestration chambers, the width W_(pr) of aproximal opening of a connection region may be at least as large as thelargest dimension of a micro-object (e.g., a biological micro-objectsuch as a cell) that the sequestration chamber is intended for. Forexample, the width W_(pr) may be about 50 microns, about 60 microns,about 100 microns, about 200 microns, about 300 microns or may be in arange of about 50-300 microns, about 50-200 microns, about 50-100microns, about 75-150 microns, about 75-100 microns, or about 200-300microns

In various embodiments of sequestration chambers, a ratio of the lengthL_(con) of a connection region (e.g., 236) to a width W_(con) of theconnection region (e.g., 236) at the proximal opening 234 can be greaterthan or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. Theforegoing are examples only, and the ratio of the length L_(con) of aconnection region 236 to a width W_(con) of the connection region 236 atthe proximal opening 234 can be different than the foregoing examples.

In various embodiments of microfluidic devices 100, 200, 23, 250, 280,290, 300, V_(max) can be set around 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 microliters/sec.

In various embodiments of microfluidic devices having sequestrationchambers, the volume of an isolation region (e.g., 240) of asequestration chamber can be, for example, at least 5×10⁵, 8×10⁵, 1×10⁶,2×10⁶, 4×10⁶, 6×10⁶, 8×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, or 8×10⁸ cubicmicrons, or more. In various embodiments of microfluidic devices havingsequestration chambers, the volume of a sequestration chamber may beabout 5×10⁵, 6×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, 4×10⁶, 8×10⁶, 1×10⁷, 3×10⁷,5×10⁷, or about 8×10⁷ cubic microns, or more. In some other embodiments,the volume of a sequestration chamber may be about 1 nanoliter to about50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters toabout 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about2 nanoliters to about 10 nanoliters.

In various embodiment, the microfluidic device has sequestrationchambers configured as in any of the embodiments discussed herein wherethe microfluidic device has about 5 to about 10 sequestration chambers,about 10 to about 50 sequestration chambers, about 100 to about 500sequestration chambers; about 200 to about 1000 sequestration chambers,about 500 to about 1500 sequestration chambers, about 1000 to about 2000sequestration chambers, or about 1000 to about 3500 sequestrationchambers. The sequestration chambers need not all be the same size andmay include a variety of configurations (e.g., different widths,different features within the sequestration chamber.

FIG. 2G illustrates a microfluidic device 280 according to oneembodiment. The microfluidic device 280 is illustrated in FIG. 2G is astylized diagram of a microfluidic device 100. In practice themicrofluidic device 280 and its constituent circuit elements (e.g.channels 122 and sequestration chambers 128) would have the dimensionsdiscussed herein. The microfluidic circuit 120 illustrated in FIG. 2Ghas two ports 107, four distinct channels 122 and four distinct flowpaths 106. The microfluidic device 280 further comprises a plurality ofsequestration chambers opening off of each channel 122. In themicrofluidic device illustrated in FIG. 2G, the sequestration chambershave a geometry similar to the pens illustrated in FIG. 2C and thus,have both connection regions and isolation regions. Accordingly, themicrofluidic circuit 120 includes both swept regions (e.g. channels 122and portions of the connection regions 236 within the maximumpenetration depth D_(p) of the secondary flow 244) and non-swept regions(e.g. isolation regions 240 and portions of the connection regions 236not within the maximum penetration depth D_(p) of the secondary flow244).

E. System Nest

FIGS. 3A through 3B shows various embodiments of system 150 which can beused to operate and observe microfluidic devices (e.g. 100, 200, 230,250, 280, 290, 300) according to the present disclosure. As illustratedin FIG. 3A, the system 150 can include a structure (“nest”) 300configured to hold a microfluidic device 100 (not shown), or any othermicrofluidic device described herein. The nest 300 can include a socket302 capable of interfacing with the microfluidic device 320 (e.g., anoptically-actuated electrokinetic device 100) and providing electricalconnections from power source 192 to microfluidic device 320. The nest300 can further include an integrated electrical signal generationsubsystem 304. The electrical signal generation subsystem 304 can beconfigured to supply a biasing voltage to socket 302 such that thebiasing voltage is applied across a pair of electrodes in themicrofluidic device 320 when it is being held by socket 302. Thus, theelectrical signal generation subsystem 304 can be part of power source192. The ability to apply a biasing voltage to microfluidic device 320does not mean that a biasing voltage will be applied at all times whenthe microfluidic device 320 is held by the socket 302. Rather, in mostcases, the biasing voltage will be applied intermittently, e.g., only asneeded to facilitate the generation of electrokinetic forces, such asdielectrophoresis or electro-wetting, in the microfluidic device 320.

As illustrated in FIG. 3A, the nest 300 can include a printed circuitboard assembly (PCBA) 322. The electrical signal generation subsystem304 can be mounted on and electrically integrated into the PCBA 322. Theexemplary support includes socket 302 mounted on PCBA 322, as well.

Typically, the electrical signal generation subsystem 304 will include awaveform generator (not shown). The electrical signal generationsubsystem 304 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 320 held by the socket 302. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 320 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 300 further comprises a controller 308,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 304. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 308 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1A) toperform functions and analysis. In the embodiment illustrated in FIG. 3Athe controller 308 communicates with a master controller 154 through aninterface 310 (e.g., a plug or connector).

In some embodiments, the nest 300 can comprise an electrical signalgeneration subsystem 304 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya unit is configured to measurethe amplified voltage at the microfluidic device 320 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 320 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 322,resulting in a signal of up to 13 Vpp at the microfluidic device 100.

As illustrated in FIG. 3A, the support structure 300 (e.g., nest) canfurther include a thermal control subsystem 306. The thermal controlsubsystem 306 can be configured to regulate the temperature ofmicrofluidic device 320 held by the support structure 300. For example,the thermal control subsystem 306 can include a Peltier thermoelectricdevice (not shown) and a cooling unit (not shown). The Peltierthermoelectric device can have a first surface configured to interfacewith at least one surface of the microfluidic device 320. The coolingunit can be, for example, a cooling block (not shown), such as aliquid-cooled aluminum block. A second surface of the Peltierthermoelectric device (e.g., a surface opposite the first surface) canbe configured to interface with a surface of such a cooling block. Thecooling block can be connected to a fluidic path 314 configured tocirculate cooled fluid through the cooling block. In the embodimentillustrated in FIG. 3A, the support structure 300 comprises an inlet 316and an outlet 318 to receive cooled fluid from an external reservoir(not shown), introduce the cooled fluid into the fluidic path 314 andthrough the cooling block, and then return the cooled fluid to theexternal reservoir. In some embodiments, the Peltier thermoelectricdevice, the cooling unit, and/or the fluidic path 314 can be mounted ona casing 312 of the support structure 300. In some embodiments, thethermal control subsystem 306 is configured to regulate the temperatureof the Peltier thermoelectric device so as to achieve a targettemperature for the microfluidic device 320. Temperature regulation ofthe Peltier thermoelectric device can be achieved, for example, by athermoelectric power supply, such as a Pololu™ thermoelectric powersupply (Pololu Robotics and Electronics Corp.). The thermal controlsubsystem 306 can include a feedback circuit, such as a temperaturevalue provided by an analog circuit. Alternatively, the feedback circuitcan be provided by a digital circuit.

In some embodiments, the nest 300 can include a thermal controlsubsystem 306 with a feedback circuit that is an analog voltage dividercircuit (not shown) which includes a resistor (e.g., with resistance 1kOhm+/−0.1%, temperature coefficient+/−0.02 ppm/CO) and a NTC thermistor(e.g., with nominal resistance 1 kOhm+/−0.01%). In some instances, thethermal control subsystem 306 measures the voltage from the feedbackcircuit and then uses the calculated temperature value as input to anon-board PID control loop algorithm. Output from the PID control loopalgorithm can drive, for example, both a directional and apulse-width-modulated signal pin on a Pololu™ motor drive (not shown) toactuate the thermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 300 can include a serial port 324 which allows themicroprocessor of the controller 308 to communicate with an externalmaster controller 154 via the interface. In addition, the microprocessorof the controller 308 can communicate (e.g., via a Plink tool (notshown)) with the electrical signal generation subsystem 304 and thermalcontrol subsystem 306. Thus, via the combination of the controller 308,the interface 310, and the serial port 324, the electrical signalgeneration subsystem 304 and the thermal control subsystem 306 cancommunicate with the external master controller 154. In this manner, themaster controller 154 can, among other things, assist the electricalsignal generation subsystem 304 by performing scaling calculations foroutput voltage adjustments. A Graphical User Interface (GUI) (not shown)provided via a display device 170 coupled to the external mastercontroller 154, can be configured to plot temperature and waveform dataobtained from the thermal control subsystem 306 and the electricalsignal generation subsystem 304, respectively. Alternatively, or inaddition, the GUI can allow for updates to the controller 308, thethermal control subsystem 306, and the electrical signal generationsubsystem 304.

F. System Imaging Device

As discussed above, system 150 can include an imaging device. In someembodiments, the imaging device comprises a light modulating subsystem330 (See FIG. 3B). The light modulating subsystem 330 can include adigital mirror device (DMD) or a microshutter array system (MSA), eitherof which can be configured to receive light from a light source 332 andtransmits a subset of the received light into an optical train ofmicroscope 350. Alternatively, the light modulating subsystem 330 caninclude a device that produces its own light (and thus dispenses withthe need for a light source 332), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 330 can be, for example, a projector. Thus, the lightmodulating subsystem 330 can be capable of emitting both structured andunstructured light. One example of a suitable light modulating subsystem330 is the Mosaic™ system from Andor Technologies™. In certainembodiments, imaging module 164 and/or motive module 162 of system 150can control the light modulating subsystem 330.

In certain embodiments, the imaging device further comprises amicroscope 350. In such embodiments, the nest 300 and light modulatingsubsystem 330 can be individually configured to be mounted on themicroscope 350. The microscope 350 can be, for example, a standardresearch-grade light microscope or fluorescence microscope. Thus, thenest 300 can be configured to be mounted on the stage 344 of themicroscope 350 and/or the light modulating subsystem 330 can beconfigured to mount on a port of microscope 350. In other embodiments,the nest 300 and the light modulating subsystem 330 described herein canbe integral components of microscope 350.

In certain embodiments, the microscope 350 can further include one ormore detectors 348. In some embodiments, the detector 348 is controlledby the imaging module 164. The detector 348 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 348 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope350 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 320 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 348. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device is configured to use at least twolight sources. For example, a first light source 332 can be used toproduce structured light (e.g., via the light modulating subsystem 330)and a second light source 334 can be used to provide unstructured light.The first light source 332 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 334 can be used to provide bright fieldillumination. In these embodiments, the motive module 164 can be used tocontrol the first light source 332 and the imaging module 164 can beused to control the second light source 334. The optical train of themicroscope 350 can be configured to (1) receive structured light fromthe light modulating subsystem 330 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device, when the device is being heldby the nest 300, and (2) receive reflected and/or emitted light from themicrofluidic device and focus at least a portion of such reflectedand/or emitted light onto detector 348. The optical train can be furtherconfigured to receive unstructured light from a second light source andfocus the unstructured light on at least a second region of themicrofluidic device, when the device is held by the nest 300. In certainembodiments, the first and second regions of the microfluidic device canbe overlapping regions. For example, the first region can be a subset ofthe second region. In other embodiments, the second light source 334 mayadditionally or alternatively include a laser, which may have anysuitable wavelength of light. The representation of the optical systemshown in FIG. 3B is a schematic representation only, and the opticalsystem may include additional filters, notch filters, lenses and thelike. When the second light source 334 includes one or more lightsource(s) for brightfield and/or fluorescent excitation, as well aslaser illumination the physical arrangement of the light source(s) mayvary from that shown in FIG. 3B, and the laser illumination may beintroduced at any suitable physical location within the optical system.The schematic locations of light source 432 and light source 402/lightmodulating subsystem 404 may be interchanged as well.

In FIG. 3B, the first light source 332 is shown supplying light to alight modulating subsystem 330, which provides structured light to theoptical train of the microscope 350 of system 355 (not shown). Thesecond light source 334 is shown providing unstructured light to theoptical train via a beam splitter 336. Structured light from the lightmodulating subsystem 330 and unstructured light from the second lightsource 334 travel from the beam splitter 336 through the optical traintogether to reach a second beam splitter (or dichroic filter 338,depending on the light provided by the light modulating subsystem 330),where the light gets reflected down through the objective 336 to thesample plane 342. Reflected and/or emitted light from the sample plane342 then travels back up through the objective 340, through the beamsplitter and/or dichroic filter 338, and to a dichroic filter 346. Onlya fraction of the light reaching dichroic filter 346 passes through andreaches the detector 348.

In some embodiments, the second light source 334 emits blue light. Withan appropriate dichroic filter 346, blue light reflected from the sampleplane 342 is able to pass through dichroic filter 346 and reach thedetector 348. In contrast, structured light coming from the lightmodulating subsystem 330 gets reflected from the sample plane 342, butdoes not pass through the dichroic filter 346. In this example, thedichroic filter 346 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 330 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 330 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 346 to reach the detector348. In such an embodiment, the filter 346 acts to change the balancebetween the amount of light that reaches the detector 348 from the firstlight source 332 and the second light source 334. This can be beneficialif the first light source 332 is significantly stronger than the secondlight source 334. In other embodiments, the second light source 334 canemit red light, and the dichroic filter 346 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

G. Coating Solutions and Coating Agents.

Without intending to be limited by theory, maintenance of a biologicalmicro-object (e.g., a biological cell) within a microfluidic device(e.g., a DEP-configured and/or EW-configured microfluidic device) may befacilitated (i.e., the biological micro-object exhibits increasedviability, greater expansion and/or greater portability within themicrofluidic device) when at least one or more inner surfaces of themicrofluidic device have been conditioned or coated so as to present alayer of organic and/or hydrophilic molecules that provides the primaryinterface between the microfluidic device and biological micro-object(s)maintained therein. In some embodiments, one or more of the innersurfaces of the microfluidic device (e.g. the inner surface of theelectrode activation substrate of a DEP-configured microfluidic device,the cover of the microfluidic device, and/or the surfaces of the circuitmaterial) may be treated with or modified by a coating solution and/orcoating agent to generate the desired layer of organic and/orhydrophilic molecules.

The coating may be applied before or after introduction of biologicalmicro-object(s), or may be introduced concurrently with the biologicalmicro-object(s). In some embodiments, the biological micro-object(s) maybe imported into the microfluidic device in a fluidic medium thatincludes one or more coating agents. In other embodiments, the innersurface(s) of the microfluidic device (e.g., a DEP-configuredmicrofluidic device) are treated or “primed” with a coating solutioncomprising a coating agent prior to introduction of the biologicalmicro-object(s) into the microfluidic device.

In some embodiments, at least one surface of the microfluidic deviceincludes a coating material that provides a layer of organic and/orhydrophilic molecules suitable for maintenance and/or expansion ofbiological micro-object(s) (e.g. provides a conditioned surface asdescribed below). In some embodiments, substantially all the innersurfaces of the microfluidic device include the coating material. Thecoated inner surface(s) may include the surface of a flow region (e.g.,channel), chamber, or sequestration chamber, or a combination thereof.In some embodiments, each of a plurality of sequestration chambers hasat least one inner surface coated with coating materials. In otherembodiments, each of a plurality of flow regions or channels has atleast one inner surface coated with coating materials. In someembodiments, at least one inner surface of each of a plurality ofsequestration chambers and each of a plurality of channels is coatedwith coating materials.

Coating Agent/Solution.

Any convenient coating agent/coating solution can be used, including butnot limited to: serum or serum factors, bovine serum albumin (BSA),polymers, detergents, enzymes, and any combination thereof.

Polymer-Based Coating Materials.

The at least one inner surface may include a coating material thatcomprises a polymer. The polymer may be covalently or non-covalentlybound (or may be non-specifically adhered) to the at least one surface.The polymer may have a variety of structural motifs, such as found inblock polymers (and copolymers), star polymers (star copolymers), andgraft or comb polymers (graft copolymers), all of which may be suitablefor the methods disclosed herein.

The polymer may include a polymer including alkylene ether moieties. Awide variety of alkylene ether containing polymers may be suitable foruse in the microfluidic devices described herein. One non-limitingexemplary class of alkylene ether containing polymers are amphiphilicnonionic block copolymers which include blocks of polyethylene oxide(PEO) and polypropylene oxide (PPO) subunits in differing ratios andlocations within the polymer chain. Pluronic® polymers (BASF) are blockcopolymers of this type and are known in the art to be suitable for usewhen in contact with living cells. The polymers may range in averagemolecular mass M, from about 2000 Da to about 20KDa. In someembodiments, the PEO-PPO block copolymer can have ahydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18).Specific Pluronic® polymers useful for yielding a coated surface includePluronic® L44, L64, P85, and F127 (including F127NF). Another class ofalkylene ether containing polymers is polyethylene glycol (PEGM_(w)<100,000 Da) or alternatively polyethylene oxide (PEO,M_(w)>100,000). In some embodiments, a PEG may have an M_(w) of about1000 Da, 5000 Da, 10,000 Da or 20,000 Da. In other embodiments, thecoating material may include a polymer containing carboxylic acidmoieties. The carboxylic acid subunit may be an alkyl, alkenyl oraromatic moiety containing subunit. One non-limiting example ispolylactic acid (PLA). In other embodiments, the coating material mayinclude a polymer containing phosphate moieties, either at a terminus ofthe polymer backbone or pendant from the backbone of the polymer. In yetother embodiments, the coating material may include a polymer containingsulfonic acid moieties. The sulfonic acid subunit may be an alkyl,alkenyl or aromatic moiety containing subunit. One non-limiting exampleis polystyrene sulfonic acid (PSSA) or polyanethole sulfonic acid. Infurther embodiments, the coating material may include a polymerincluding amine moieties. The polyamino polymer may include a naturalpolyamine polymer or a synthetic polyamine polymer. Examples of naturalpolyamines include spermine, spermidine, and putrescine.

In other embodiments, the coating material may include a polymercontaining saccharide moieties. In a non-limiting example,polysaccharides such as xanthan gum or dextran may be suitable to form amaterial which may reduce or prevent cell sticking in the microfluidicdevice. For example, a dextran polymer having a size about 3 kDa may beused to provide a coating material for a surface within a microfluidicdevice.

In other embodiments, the coating material may include a polymercontaining nucleotide moieties, i.e. a nucleic acid, which may haveribonucleotide moieties or deoxyribonucleotide moieties, providing apolyelectrolyte surface. The nucleic acid may contain only naturalnucleotide moieties or may contain unnatural nucleotide moieties whichcomprise nucleobase, ribose or phosphate moiety analogs such as7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moietieswithout limitation.

In yet other embodiments, the coating material may include a polymercontaining amino acid moieties. The polymer containing amino acidmoieties may include a natural amino acid containing polymer or anunnatural amino acid containing polymer, either of which may include apeptide, a polypeptide or a protein. In one non-limiting example, theprotein may be bovine serum albumin (BSA) and/or serum (or a combinationof multiple different sera) comprising albumin and/or one or more othersimilar proteins as coating agents. The serum can be from any convenientsource, including but not limited to fetal calf serum, sheep serum, goatserum, horse serum, and the like. In certain embodiments, BSA in acoating solution is present in a range of form about 1 mg/mL to about100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more or anywhere inbetween. In certain embodiments, serum in a coating solution may bepresent in a range of from about 20% (v/v) to about 50% v/v, including25%, 30%, 35%, 40%, 45%, or more or anywhere in between. In someembodiments, BSA may be present as a coating agent in a coating solutionat 5 mg/mL, whereas in other embodiments, BSA may be present as acoating agent in a coating solution at 70 mg/mL. In certain embodiments,serum is present as a coating agent in a coating solution at 30%. Insome embodiments, an extracellular matrix (ECM) protein may be providedwithin the coating material for optimized cell adhesion to foster cellgrowth. A cell matrix protein, which may be included in a coatingmaterial, can include, but is not limited to, a collagen, an elastin, anRGD-containing peptide (e.g. a fibronectin), or a laminin. In yet otherembodiments, growth factors, cytokines, hormones or other cell signalingspecies may be provided within the coating material of the microfluidicdevice.

In some embodiments, the coating material may include a polymercontaining more than one of alkylene oxide moieties, carboxylic acidmoieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, or amino acid moieties. In otherembodiments, the polymer conditioned surface may include a mixture ofmore than one polymer each having alkylene oxide moieties, carboxylicacid moieties, sulfonic acid moieties, phosphate moieties, saccharidemoieties, nucleotide moieties, and/or amino acid moieties, which may beindependently or simultaneously incorporated into the coating material.

Covalently Linked Coating Materials.

In some embodiments, the at least one inner surface includes covalentlylinked molecules that provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) within the microfluidic device, providing a conditionedsurface for such cells.

The covalently linked molecules include a linking group, wherein thelinking group is covalently linked to one or more surfaces of themicrofluidic device, as described below. The linking group is alsocovalently linked to a moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s).

In some embodiments, the covalently linked moiety configured to providea layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) may include alkyl orfluoroalkyl (which includes perfluoroalkyl) moieties; mono- orpolysaccharides (which may include but is not limited to dextran);alcohols (including but not limited to propargyl alcohol); polyalcohols,including but not limited to polyvinyl alcohol; alkylene ethers,including but not limited to polyethylene glycol; polyelectrolytes(including but not limited to polyacrylic acid or polyvinyl phosphonicacid); amino groups (including derivatives thereof, such as, but notlimited to alkylated amines, hydroxyalkylated amino group, guanidinium,and heterocylic groups containing an unaromatized nitrogen ring atom,such as, but not limited to morpholinyl or piperazinyl); carboxylicacids including but not limited to propiolic acid (which may provide acarboxylate anionic surface); phosphonic acids, including but notlimited to ethynyl phosphonic acid (which may provide a phosphonateanionic surface); sulfonate anions; carboxybetaines; sulfobetaines;sulfamic acids; or amino acids.

In various embodiments, the covalently linked moiety configured toprovide a layer of organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice may include non-polymeric moieties such as an alkyl moiety, asubstituted alkyl moiety, such as a fluoroalkyl moiety (including butnot limited to a perfluoroalkyl moiety), amino acid moiety, alcoholmoiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety,sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.Alternatively, the covalently linked moiety may include polymericmoieties, which may be any of the moieties described above.

In some embodiments, the covalently linked alkyl moiety may comprisescarbon atoms forming a linear chain (e.g., a linear chain of at least 10carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be anunbranched alkyl moiety. In some embodiments, the alkyl group mayinclude a substituted alkyl group (e.g., some of the carbons in thealkyl group can be fluorinated or perfluorinated). In some embodiments,the alkyl group may include a first segment, which may include aperfluoroalkyl group, joined to a second segment, which may include anon-substituted alkyl group, where the first and second segments may bejoined directly or indirectly (e.g., by means of an ether linkage). Thefirst segment of the alkyl group may be located distal to the linkinggroup, and the second segment of the alkyl group may be located proximalto the linking group.

In other embodiments, the covalently linked moiety may include at leastone amino acid, which may include more than one type of amino acid.Thus, the covalently linked moiety may include a peptide or a protein.In some embodiments, the covalently linked moiety may include an aminoacid which may provide a zwitterionic surface to support cell growth,viability, portability, or any combination thereof.

In other embodiments, the covalently linked moiety may include at leastone alkylene oxide moiety, and may include any alkylene oxide polymer asdescribed above. One useful class of alkylene ether containing polymersis polyethylene glycol (PEG M_(w)<100,000 Da) or alternativelypolyethylene oxide (PEO, M_(w)>100,000). In some embodiments, a PEG mayhave an M_(w) of about 1000 Da, 5000 Da, 10,000 Da or 20,000 Da.

The covalently linked moiety may include one or more saccharides. Thecovalently linked saccharides may be mono-, di-, or polysaccharides. Thecovalently linked saccharides may be modified to introduce a reactivepairing moiety which permits coupling or elaboration for attachment tothe surface. Exemplary reactive pairing moieties may include aldehyde,alkyne or halo moieties. A polysaccharide may be modified in a randomfashion, wherein each of the saccharide monomers may be modified or onlya portion of the saccharide monomers within the polysaccharide aremodified to provide a reactive pairing moiety that may be coupleddirectly or indirectly to a surface. One exemplar may include a dextranpolysaccharide, which may be coupled indirectly to a surface via anunbranched linker.

The covalently linked moiety may include one or more amino groups. Theamino group may be a substituted amine moiety, guanidine moiety,nitrogen-containing heterocyclic moiety or heteroaryl moiety. The aminocontaining moieties may have structures permitting pH modification ofthe environment within the microfluidic device, and optionally, withinthe sequestration chambers and/or flow regions (e.g., channels).

The coating material providing a conditioned surface may comprise onlyone kind of covalently linked moiety or may include more than onedifferent kind of covalently linked moiety. For example, the fluoroalkylconditioned surfaces (including perfluoroalkyl) may have a plurality ofcovalently linked moieties which are all the same, e.g., having the samelinking group and covalent attachment to the surface, the same overalllength, and the same number of fluoromethylene units comprising thefluoroalkyl moiety. Alternatively, the coating material may have morethan one kind of covalently linked moiety attached to the surface. Forexample, the coating material may include molecules having covalentlylinked alkyl or fluoroalkyl moieties having a specified number ofmethylene or fluoromethylene units and may further include a further setof molecules having charged moieties covalently attached to an alkyl orfluoroalkyl chain having a greater number of methylene orfluoromethylene units, which may provide capacity to present bulkiermoieties at the coated surface. In this instance, the first set ofmolecules having different, less sterically demanding termini and fewerbackbone atoms can help to functionalize the entire substrate surfaceand thereby prevent undesired adhesion or contact with thesilicon/silicon oxide, hafnium oxide or alumina making up the substrateitself. In another example, the covalently linked moieties may provide azwitterionic surface presenting alternating charges in a random fashionon the surface.

Conditioned Surface Properties.

Aside from the composition of the conditioned surface, other factorssuch as physical thickness of the hydrophobic material can impact DEPforce. Various factors can alter the physical thickness of theconditioned surface, such as the manner in which the conditioned surfaceis formed on the substrate (e.g. vapor deposition, liquid phasedeposition, spin coating, flooding, and electrostatic coating). In someembodiments, the conditioned surface has a thickness in the range ofabout 1 nm to about 10 nm; about 1 nm to about 7 nm; about 1 nm to about5 nm; or any individual value therebetween. In other embodiments, theconditioned surface formed by the covalently linked moieties may have athickness of about 10 nm to about 50 nm. In various embodiments, theconditioned surface prepared as described herein has a thickness of lessthan 10 nm. In some embodiments, the covalently linked moieties of theconditioned surface may form a monolayer when covalently linked to thesurface of the microfluidic device (e.g., a DEP configured substratesurface) and may have a thickness of less than 10 nm (e.g., less than 5nm, or about 1.5 to 3.0 nm). These values are in contrast to that of aCYTOP® (Asahi Glass Co., Ltd. JP) fluoropolymer spin coating, which hasa thickness in the range of about 30 nm. In some embodiments, theconditioned surface does not require a perfectly formed monolayer to besuitably functional for operation within a DEP-configured microfluidicdevice.

In various embodiments, the coating material providing a conditionedsurface of the microfluidic device may provide desirable electricalproperties. Without intending to be limited by theory, one factor thatimpacts robustness of a surface coated with a particular coatingmaterial is intrinsic charge trapping. Different coating materials maytrap electrons, which can lead to breakdown of the coating material.Defects in the coating material may increase charge trapping and lead tofurther breakdown of the coating material. Similarly, different coatingmaterials have different dielectric strengths (i.e. the minimum appliedelectric field that results in dielectric breakdown), which may impactcharge trapping. In certain embodiments, the coating material can havean overall structure (e.g., a densely-packed monolayer structure) thatreduces or limits that amount of charge trapping.

In addition to its electrical properties, the conditioned surface mayalso have properties that are beneficial in use with biologicalmolecules. For example, a conditioned surface that contains fluorinated(or perfluorinated) carbon chains may provide a benefit relative toalkyl-terminated chains in reducing the amount of surface fouling.Surface fouling, as used herein, refers to the amount of indiscriminatematerial deposition on the surface of the microfluidic device, which mayinclude permanent or semi-permanent deposition of biomaterials such asprotein and its degradation products, nucleic acids and respectivedegradation products and the like.

Unitary or Multi-Part Conditioned Surface.

The covalently linked coating material may be formed by reaction of amolecule which already contains the moiety configured to provide a layerof organic and/or hydrophilic molecules suitable formaintenance/expansion of biological micro-object(s) in the microfluidicdevice, as is described below. Alternatively, the covalently linkedcoating material may be formed in a two-part sequence by coupling themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) to a surface modifying ligand that itself has beencovalently linked to the surface.

Methods of Preparing a Covalently Linked Coating Material.

In some embodiments, a coating material that is covalently linked to thesurface of a microfluidic device (e.g., including at least one surfaceof the sequestration chambers and/or flow regions) has a structure ofFormula 1 or Formula 2. When the coating material is introduced to thesurface in one step, it has a structure of Formula 1, while when thecoating material is introduced in a multiple step process, it has astructure of Formula 2.

The coating material may be linked covalently to oxides of the surfaceof a DEP-configured or EW-configured substrate. The DEP- orEW-configured substrate may comprise silicon, silicon oxide, alumina, orhafnium oxide. Oxides may be present as part of the native chemicalstructure of the substrate or may be introduced as discussed below.

The coating material may be attached to the oxides via a linking group(“LG”), which may be a siloxy or phosphonate ester group formed from thereaction of a siloxane or phosphonic acid group with the oxides. Themoiety configured to provide a layer of organic and/or hydrophilicmolecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device can be any of the moietiesdescribed herein. The linking group LG may be directly or indirectlyconnected to the moiety configured to provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s) in the microfluidic device. When the linking group LG isdirectly connected to the moiety, optional linker (“L”) is not presentand n is 0. When the linking group LG is indirectly connected to themoiety, linker L is present and n is 1. The linker L may have a linearportion where a backbone of the linear portion may include 1 to 200non-hydrogen atoms selected from any combination of silicon, carbon,nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemicalbonding limitations as is known in the art. It may be interrupted withany combination of one or more moieties selected from the group ofether, ammo, carbonyl, amido, or phosphonate groups, arylene,heteroarylene, or heterocyclic groups. In some embodiments, the backboneof the linker L may include 10 to 20 atoms. In other embodiments, thebackbone of the linker L may include about 5 atoms to about 200 atoms;about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; orabout 10 atoms to about 40 atoms. In some embodiments, the backboneatoms are all carbon atoms.

In some embodiments, the moiety configured to provide a layer of organicand/or hydrophilic molecules suitable for maintenance/expansion ofbiological micro-object(s) may be added to the surface of the substratein a multi-step process, and has a structure of Formula 2, as shownabove. The moiety may be any of the moieties described above.

In some embodiments, the coupling group CG represents the resultantgroup from reaction of a reactive moiety RX and a reactive pairingmoiety Rpx (i.e., a moiety configured to react with the reactive moietyRX). For example, one typical coupling group CG may include acarboxamidyl group, which is the result of the reaction of an aminogroup with a derivative of a carboxylic acid, such as an activatedester, an acid chloride or the like. Other CG may include a triazolylenegroup, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide,an ether, or alkenyl group, or any other suitable group that may beformed upon reaction of a reactive moiety with its respective reactivepairing moiety. The coupling group CG may be located at the second end(i.e., the end proximal to the moiety configured to provide a layer oforganic and/or hydrophilic molecules suitable for maintenance/expansionof biological micro-object(s) in the microfluidic device) of linker L,which may include any combination of elements as described above. Insome other embodiments, the coupling group CG may interrupt the backboneof the linker L. When the coupling group CG is triazolylene, it may bethe product resulting from a Click coupling reaction and may be furthersubstituted (e.g., a dibenzocylcooctenyl fused triazolylene group).

In some embodiments, the coating material (or surface modifying ligand)is deposited on the inner surfaces of the microfluidic device usingchemical vapor deposition. The vapor deposition process can beoptionally improved, for example, by pre-cleaning the cover 110, themicrofluidic circuit material 116, and/or the substrate (e.g., the innersurface 208 of the electrode activation substrate 206 of aDEP-configured substrate, or a dielectric layer of the support structure104 of an EW-configured substrate), by exposure to a solvent bath,sonication or a combination thereof. Alternatively, or in addition, suchpre-cleaning can include treating the cover 110, the microfluidiccircuit material 116, and/or the substrate in an oxygen plasma cleaner,which can remove various impurities, while at the same time introducingan oxidized surface (e.g. oxides at the surface, which may be covalentlymodified as described herein). Alternatively, liquid-phase treatments,such as a mixture of hydrochloric acid and hydrogen peroxide or amixture of sulfuric acid and hydrogen peroxide (e.g., piranha solution,which may have a ratio of sulfuric acid to hydrogen peroxide in a rangefrom about 3:1 to about 7:1) may be used in place of an oxygen plasmacleaner.

In some embodiments, vapor deposition is used to coat the inner surfacesof the microfluidic device 200 after the microfluidic device 200 hasbeen assembled to form an enclosure 102 defining a microfluidic circuit120. Without intending to be limited by theory, depositing such acoating material on a fully-assembled microfluidic circuit 120 may bebeneficial in preventing delamination caused by a weakened bond betweenthe microfluidic circuit material 116 and the electrode activationsubstrate 206 dielectric layer and/or the cover 110. In embodimentswhere a two-step process is employed the surface modifying ligand may beintroduced via vapor deposition as described above, with subsequentintroduction of the moiety configured provide a layer of organic and/orhydrophilic molecules suitable for maintenance/expansion of biologicalmicro-object(s). The subsequent reaction may be performed by exposingthe surface modified microfluidic device to a suitable coupling reagentin solution.

FIG. 2H depicts a cross-sectional view of a microfluidic device 290having an exemplary covalently linked coating material providing aconditioned surface. As illustrated, the coating materials 298 (shownschematically) can comprise a monolayer of densely-packed moleculescovalently bound to both the inner surface 294 of a base 286, which maybe a DEP substrate, and the inner surface 292 of a cover 288 of themicrofluidic device 290. The coating material 298 can be disposed onsubstantially all inner surfaces 294, 292 proximal to, and facinginwards towards, the enclosure 284 of the microfluidic device 290,including, in some embodiments and as discussed above, the surfaces ofmicrofluidic circuit material (not shown) used to define circuitelements and/or structures within the microfluidic device 290. Inalternate embodiments, the coating material 298 can be disposed on onlyone or some of the inner surfaces of the microfluidic device 290.

In the embodiment shown in FIG. 2H, the coating material 298 can includea monolayer of organosiloxane molecules, each molecule covalently bondedto the inner surfaces 292, 294 of the microfluidic device 290 via asiloxy linker 296. Any of the above-discussed coating materials 298 canbe used (e.g. an alkyl-terminated, a fluoroalkyl terminated moiety, aPEG-terminated moiety, a dextran terminated moiety, or a terminal moietycontaining positive or negative charges for the organosiloxy moieties),where the terminal moiety is disposed at its enclosure-facing terminus(i.e. the portion of the monolayer of the coating material 298 that isnot bound to the inner surfaces 292, 294 and is proximal to theenclosure 284).

In other embodiments, the coating material 298 used to coat the innersurface(s) 292, 294 of the microfluidic device 290 can include anionic,cationic, or zwitterionic moieties, or any combination thereof. Withoutintending to be limited by theory, by presenting cationic moieties,anionic moieties, and/or zwitterionic moieties at the inner surfaces ofthe enclosure 284 of the microfluidic circuit 120, the coating material298 can form strong hydrogen bonds with water molecules such that theresulting water of hydration acts as a layer (or “shield”) thatseparates the biological micro-objects from interactions withnon-biological molecules (e.g., the silicon and/or silicon oxide of thesubstrate). In addition, in embodiments in which the coating material298 is used in conjunction with coating agents, the anions, cations,and/or zwitterions of the coating material 298 can form ionic bonds withthe charged portions of non-covalent coating agents (e.g. proteins insolution) that are present in a medium 180 (e.g. a coating solution) inthe enclosure 284.

In still other embodiments, the coating material may comprise or bechemically modified to present a hydrophilic coating agent at itsenclosure-facing terminus. In some embodiments, the coating material mayinclude an alkylene ether containing polymer, such as PEG. In someembodiments, the coating material may include a polysaccharide, such asdextran. Like the charged moieties discussed above (e.g., anionic,cationic, and zwitterionic moieties), the hydrophilic coating agent canform strong hydrogen bonds with water molecules such that the resultingwater of hydration acts as a layer (or “shield”) that separates thebiological micro-objects from interactions with non-biological molecules(e.g., the silicon and/or silicon oxide of the substrate).

Further details of appropriate coating treatments and modifications maybe found at U.S. application Ser. No. 15/135,707, filed on Apr. 22,2016, and is incorporated by reference in its entirety.

H. Additional System Components for Maintenance of Viability of Cellswithin the Sequestration Chambers of the Microfluidic Device.

In order to promote growth and/or expansion of cell populations,environmental conditions conducive to maintaining functional cells maybe provided by additional components of the system. For example, suchadditional components can provide nutrients, cell growth signalingspecies, pH modulation, gas exchange, temperature control, and removalof waste products from cells.

IV. Additional Embodiments

A. Embodiments Using T or NK Cells

In certain instances, T cells may be present in the dissociated cellsample. In such instances, the method may further comprise performing aselection on the dissociated cell sample to isolate a fraction that hasa greater percentage or concentration of T cells than the originaldissociated sample. In this manner, tumor-infiltrating lymphocytes(TILs) can be obtained from the same patient's tumor sample. In otherinstances, T cells can be obtained from the same patient's blood. Forexample, T cells can be selected from a blood sample (or peripheralblood mononuclear cell (PBMC) sample) obtained from the same patient. Instill other instances, T cells can be obtained from a subject that isnot the patient providing the at least one solid tumor (e.g., a subjectthat is a blood donor or tissue donor). T cells may be separated fromother cells in a dissociated cell sample or a blood/PBMC sample by usingat least one T cell marker chosen from CD4, CD8, CD25, CD45RA, CD45RO,CD62L, CD69, and CD3. Regardless of the origin of the T cells or theselection procedure used (if any), the resulting T cells may be clonedand/or used in the methods disclosed herein. For example, the T cells(whether patient-specific or donor-derived) may be used to prepare CAR-Ttherapeutics or used for other types of immunotherapy, such ascombination therapy involving TILs and another therapeutic agent (e.g.,an antibody prepared according to the methods disclosed herein).

Similar to T cells, natural killer (NK) cells can be used in the methodsdisclosed herein. For example, NK cells can be used to prepare CARtherapeutics, as described in Topfer et al. (2015), J. Immunol.194(7):3201-3212. The NK cells can be obtained, for example, from ablood sample or a tissue sample (e.g., a dissociated cell sampleobtained from a tumor). The NK cells can be isolated from other cells inthe sample by using at least one NK marker, such as CD56.

B. Embodiments Relying on Outside Cell Sources

In some embodiments, all of the cell sources are from a single patient.In other embodiments, such as those discussed in this subsection, thecell sources may be from different patients or different sources. In oneaspect, the method comprises using a microfluidic device to identify atleast one B cell that produces antibodies capable of binding to both thepatient's cancer cells and cancer cells from another source (e.g., in atwo-step process). In one embodiment, the B cells are tested against acancer cell line while the patient's own cancer cells are preparedand/or cultured (e.g., before the patient's cancer cells are loaded ontothe microfluidic device). T cells or NK cells may also be obtained fromthe same patient or from a different source.

C. Embodiments Employing Normal Cells

When desired, the specificity of the antibodies produced by the B cellscan be evaluated by comparing their ability to bind both cancer andnon-cancer cells (i.e., normal cells). As such, optionally, the methodmay further comprise using a microfluidic device to identify whether theantibodies produced by the B cell are capable of binding to non-cancercells. The non-cancer cells and the cancer cells may be dissociated fromthe same tumor sample. Alternatively, the non-cancer cells and/or thecancer cells may originate from a subject other than the patientproviding the at least one solid tumor sample.

The same microfluidic device may be used to identify binding of theantibodies to cancer and non-cancer cells. The cancer and non-cancercells may be loaded into different parts of the microfluidic device. Forexample, cancer cells (or non-cancer cells) can be placed into the sameisolation chamber as a B cell, and non-cancer cells (or cancer cells)can be flowed into the flow channel to a location proximal to where theisolation chamber opens into the flow channel. In another example,cancer cells (or non-cancer cells) can be placed into an isolationchamber that is adjacent to an isolation chamber that contains a B cell,and non-cancer cells (or cancer cells) can be flowed into the flowchannel to a location proximal to where the isolation chamber thatcontains the B cell opens into the flow channel. In still anotherexample, cancer cells (or non-cancer cells) can be placed into anisolation chamber along with a B cell, and non-cancer cells (or cancercells) can be placed into an isolation chamber that is adjacent to theisolation chamber that contains the B cell. Regardless of the placementof the cancer cells and/or the non-cancer cells, the same microfluidicdevice can be used to simultaneous assess whether antibodies (e.g., suchas produced by a B cell) bind to cancer cells and/or non-cancer cells.In other embodiments, the cancer and non-cancer cells may be loadedsequentially in the same flow path (or the same isolation region),allowing for the sequential detection of binding between (1) theantibodies and the cancer cells and (2) the antibodies and thenon-cancer cells.

V. Methods of Treatment and Therapeutic Compositions

Patients having cancer may be treated with an antibody or fragmentthereof produced by the methods described herein. In one instance, thetumor sample is taken from the same patient who is treated. Oneadvantage of this method is that personalized treatment can be designedfor a given patient and in some instances that personalized treatmentcan be prepared in a relatively short window of time, allowing fortherapeutic efficacy in the time required by the patient's diseasestate. For instance, in some embodiments, the time from obtaining thetumor sample to treatment may be at most about 2 months.

Once the variable heavy and light chain regions of the antibody producedby the B cell have been determined, a variety of different antibody orantibody fragment constructs may be prepared, either containing all orsome of the variable sequences. In some instances, the patient may betreated with a single chain antibody. In other instances, the patientmay be treated with an antibody or fragment thereof that has two heavychains and two light chains. In some embodiments an engineered antibodyconstruct comprises Fab or Fab′(2) fragments, scFvs, multivalent scFvs(e.g., diabodies or tribodies), minibodies (e.g., scFv-CH3 dimers),bispecific Abs, or camel variable functional heavy chain domains.

In other embodiments an engineered antibody construct comprises anyvariant of the antibody identified by the methods herein. In someembodiments, the antibody or fragment may be administered in isolatedform.

For example, an engineered antibody construct may comprise (a) at leastthe heavy chain CDRs of an antibody identified by the method herein; (b)at least the heavy and light chain CDRs of an antibody identified by themethod herein; (c) at least the heavy chain variable region of anantibody identified by the method herein; or (d) at least the heavy andlight chain variable regions of an antibody identified by the methodherein.

In other instances, various constructs may be prepared using theantibody or fragment. For instance, the antibody may be displayed on anengineered T cell or an engineered NK cell. In some embodiments, theengineered T cell is a chimeric antigen receptor T cell. The T cell maybe obtained from the same patient prior to being engineered to displaythe antibody, such as, for instance from the patient's tumor sample or ablood sample obtained from the patient. Such a T cell may be geneticallyengineered to express the antibody or fragment thereof. Similarly, inother embodiments, the engineered NK cell is a chimeric antigen receptorNK cell. The NK cell may be obtained from the same patient prior to beengineered to display the antibody, such as, for instance, from thepatient's tumor sample or a blood sample obtained from the patient. Sucha NK cell may be genetically engineered to express the antibody orfragment thereof.

In some modes, the antibody or fragment thereof may be administered incombination with another therapy. In such a case, the combinationtherapy may be surgery, radiation, chemotherapy, CAR-T therapy, T celltherapy (such as by simply amplifying the patients own T cells, such asTILs, and reintroducing them), other immunotherapy (by targeting knownantigens that inhibit T cell function with function-blocking antibodies,such as inhibitory antibodies against PD-1, PD-L1, CTLA-4, LAG-3, TIM-3,VISTA, BTLA, or any combination thereof), or administration of an immunestimulatory agent, such as ICOS, OX40, 41BB, an anti-tumor vaccine,cytokines, or a tumor-specific virus.

VI. Methods of Labeling and Detection

Antibodies produced according to the methods herein may also be used inmethods of detection. For example, the antibodies may be labeled with adetectable label and used in vivo or in vitro to detect or label cancercells (including the patient's own tumor cells). For example,fluorescence guided surgery may be performed using afluorophore-conjugated antibody in order to highlight the tumor, improvesurgical resection, and increase survival rates. Fluorophores such asAlexa Fluor 488 may be used. The antibody-fluorophore conjugate may beinjected intravenously or it may be applied to the resection surfacesduring surgery. The antibodies produced herein may also be used forother methods of detection or labeling.

EXAMPLES Example 1. Processing of Tumor Biopsy and Fragmentation ofDissociated Cell Sample

A. Dissociation of Tumor Sample

A tumor sample is obtained from a patient having a surgical resection ofhis or her tumor. On the day of tumor resection, the specimen isreceived in the laboratory immediately following the procedure. Thespecimen is bathed in sterile saline in a sterile container. Viabletumor tissue is dissected away from non-viable tissue and healthy(non-cancer) tissue in a laminar flow hood using a scalpel and sterileforceps. Other samples of viable tumor tissue may be obtained for otherassessments. The tumor sample for dissociation is weighed and kept in a50 ml centrifuge tube containing a small amount of sterile HBSS. Amedium for culturing cells is prepared by supplementing RPMI 1640 with10% human serum (heat inactivated at 56° C. for 30 minutes), and withfinal concentrations of penicillin G (100 units/ml), streptomycin (100μg/ml), gentamicin (50 μg/ml), Hepes (25 mM), and 2-mercaptoethanol(5.5×10⁻⁵ M).

The tissue specimen is placed on a sterile cutting surface (cuttingboard or open Petri dish). Using sterile scalpel and forceps, thespecimen is cut into small (3-5 mm) fragments. Cut fragments aretransferred into a gentleMACS® C Tube. Enzyme medium (EM) is added tothe tube (5 ml for 0.2-2 g of tissue and 10 ml for 2-5 g) and the tubeis securely closed by turning the cap until a slight click is heard. EMis used to help disperse cells from the surgical specimen during theshort incubation periods between the gentleMACS® program runs. Theenzyme-containing medium, RPMI 1640, does not contain serum, butcontains penicillin G (100 units/ml), streptomycin (100 μg/ml),gentamicin (50 μg/ml), Fungizone® (1.25 μg/ml), collagenase (1 mg/ml),and Pulmozyme® (˜30 units/ml). Enzyme stocks are dry powders stored at−20° C.

The C Tubes are installed vertically and cap-side down into thegentleMACs Sleeve. Proper installation ensures that the C Tubes are heldin position against rotational and axial forces. Pre-defined programsare provided by the internal gentleMACS® dissociator memory. Theprograms vary in intensity, so an appropriate program is selecteddepending on the texture of the tissues to be processed. Softer tissuesrequire more gentle rotation (lower speed) and harder tissues require amore vigorous, longer rotation. Multiple dissociation runs may beperformed with evaluation of the state of dissociation being performedbetween runs. Dissociated tissue is filtered through an autoclaved wiremesh placed in an autoclaved funnel on top of a 250 ml centrifuge tube.The wire mesh is rinsed with sterile HBSS and the tube filed withadditional sterile HBSS. The dissociated tissue is then washed one timeby centrifuging 10 minutes at 1500 rpm. The supernatant is aspirated andthe pellet resuspended in a known volume of HBSS.

B. An Alternative Procedure for Tumor Dissociation

As an alternative to performing tumor dissociation using the gentleMACS®instrument, a digestion buffer may be used. Tumor tissue is teased apartusing forceps or cut into small pieces (2-5 mm). Tissue is placed in adissociation buffer (100 units/ml collagenase and 100 μg/ml DNase inRPMI and 10% human serum (heat inactivated at 56° C. for 30 minutes)).The digestion proceeds in an incubator at 37° C. for 30 minutes. Afterincubation, the sample is pipetted up and down with a pipette to get aneasily flowing single cell suspension.

The suspension is filtered through a 70 micron filter and washed 2× withMACs separation buffer supplemented with 10% human serum (heatinactivated at 56° C. for 30 minutes). The cell suspension iscentrifuged at 400×g for 10 minutes. The pellet is rinsed with 10 mlMACs buffer and centrifuged again at the same setting. The dissociatedcells are then resuspended in 100 μl MACs buffer.

C. Fragmentation of Cell Types from Dissociated Cell Sample

After a dissociated cell sample comprising single cells is preparedusing either protocol above, B cells, T cells, NK cells, and cancercells may be fragmented from the dissociated cell sample. Anti-CD20magnetic beads may be used to select for B cells. Anti-CD4 and/oranti-CD8 magnetic beads may be used to select for T cells. Anti-CD56magnetic beads may be used to select for NK cells. Magnetic beadsattached to an antibody specific for the cancer cells may be used toselect for cancer cells or cancer cells may be removed manually usingmorphological evaluations.

Antibody labeled microbeads targeting the first cell type to befragmented (approximately 10 μl per 10⁸ cells) are added to thedissociated cell sample. The tube is mixed well by gentle flicking ofthe tube and incubating for 15 minutes at 4-8° C., shielded from light.The cells are washed by adding 10 ml of MACs buffer and centrifuging at400×g for 10 minutes. The supernatant is poured (or aspirated) off andthe cells resuspended in 500 μl MACs buffer. A fresh column is appliedto the magnet separator. The column is primed by rinsing with anappropriate amount of MACs buffer for the column selected. After primingthe column, the cell suspension is applied to the top of the column,with one column used for every 1×10⁸ cells. Any unlabeled cells(pass-through) are collected and reserved for isolation of differentcell types. For a first wash, the column is washed for a minimum ofthree and up to five times with a 500 μl volume for a total of 1.5 to2.5 ml of fluid wash. Fluid flow is preserved in the column without itbeing allowed to run dry, which can impact purity and lead to loss ofcell viability).

For a second wash, the column is washed with a dissociation buffer mixcomprising collagenase and DNase in order to flush out sticky debrisfrom dead or dying tumor cells (100 units/ml Collegenase and 100 μg/mlDNase in RPMI and 10% human serum (heat inactivated at 56° C. for 30minutes)). For a third wash, the MACs buffer is used again until thewash looks completely clear.

The column is removed from the magnetic separator and placed on asuitable collection tube. 2 ml of buffer is placed on the column and themagnetically labeled cells are collected by firmly applying the plungersupplied with the column.

Alternatively, magnetic separation may be conducted by AutoMACS®.

Additional separation steps are performed to isolate each of the Bcells, T cells, NK cells, and cancer cells. Magnetic beads are removedfrom the cells, when desired.

Example 2. Screening Mouse Splenocytes for Secretion of IgG Antibodiesin a Microfluidic Device

A screen was performed to identify mouse splenocytes that secreteIgG-type antibodies that bind to human CD45. The experimental designincluded the following steps:

1. Generation of CD45 antigen coated beads;

2. Harvest mouse splenocytes;

3. Load cells into a microfluidic device; and

4. Assay for antigen specificity.

Reagents used for the experiment included those shown in Table 1.

TABLE 1 Reagents Name Vendor Catalog Number Lot Number 1 Slide-A-LyzerMINI Dialysis Thermo Pierce 69560 OJ189254 Device, 7K MWCO, 0.1 mL 2CD45 Protein R&D Systems 1430-CD 112722 3 PBS pH 7.2 with Mg2+ and Ca2+Fisher BP29404 4 Streptavidin Coated Beads (8 μm) Spherotech SVP-60-5AC01 5 EZ-Link NHS-PEG4-Biotin, No- Pierce 21329 Weigh Format 6Hybridoma SFM Media Life Tech 12045-076 7 Fetal Bovine Serum Hyclone#SH30084.03 8 Penicillin-Streptomycin (10,000 Life 15140-122 U/mL) 9Goat anti-mouse F(ab′)2-Alexa 568 Life Cat# A11019 Lot#1073003 10streptavidin-488 Life Catalog Lot #S32354 #1078760 11 Mouse anti CD45IgG₁ R&D Systems MAB1430 ILP0612061 12 BD Falcon ™ Cell Strainers, BD352340 40 μm, Blue

Generation of CD45 Antigen Coated Beads

CD45 antigen coated microbeads were generated in the following manner:

-   -   50 g carrier free CD45 was resuspended in 500 μL PBS (pH 7.2).    -   A slide-a-lyzer mini cup was rinsed with 500 μL PBS, then added        to a microfuge tube.    -   50 μL of the 0.1 μg/μL CD45 solution was added to the rinsed        slide-a-lyzer mini cup.    -   170 μL PBS was added to 2 mg of NHS-PEG4-Biotin, after which 4.1        μL of NHS-PEG4-Biotin was added to the slide-a-lyzer mini cup        containing the CD45 antigen.    -   The NGS-PEG4-Biotin was incubated with the CD45 antigen for 1        hour at room temperature.    -   Following the incubation, the slide-a-lyser mini cup was removed        from the microfuge tube, placed into 1.3 mls PBS (pH 7.2) in a        second microfuge tube, and incubated at 4° C. with rocking, for        a first 1 hour period. The slide-a-lyser mini cup was        subsequently transferred to a third microfuge tube containing        1.3 mls of fresh PBS (pH 7.2), and incubated at 4° C. with        rocking, for a second 1 hour period. This last step was repeated        three more times, for a total of five 1 hour incubations.    -   100 μL of biotinylated CD45 solution (˜50 ng/μL) was pipetted        into labeled tubes.    -   500 μL Spherotech streptavidin coated beads were pipetted into a        microfuge tube, washed 3 times (1000 μL/wash) in PBS (pH 7.4),        then centrifuges for 5 min at 3000 RCF.    -   The beads were resuspended in 500 μl PBS (pH 7.4), resulting in        a bead concentration of 5 mg/ml.    -   50 μL biotinylated protein was mixed with the resuspended        Spherotech streptavidin coated beads. The mixture was incubated        at 4° C., with rocking, for 2 hours, then centrifuged 4° for 5        min at 3000 RCF. The supernatant was discarded and the CD45        coated beads were washed 3 times in 1 mL PBS (pH 7.4). The beads        were then centrifuges at 4° C. for another 5 min at 3000 RCF.        Finally, the CD45 beads were resuspended in 500 μL PBS pH 7.4        and stored at 4° C.

Mouse Splenocyte Harvest

The spleen from a mouse immunized with CD45 was harvested and placedinto DMEM media+10% FBS. Scissors were used to mince the spleen.

Minced spleen was placed into a 40 μm cell strainer. Single cells werewashed through the cell strainer with a 10 ml pipette. A glass rod wasused to break up the spleen further and force single cells through thecell strainer, after which single cells were again washed through thecell strainer with a 10 ml pipette.

Red blood cells were lysed with a commercial kit.

Cells were spun down at 200×G and raw splenocytes were resuspended inDMEM media+10% FBS with 10 ml pipette at a concentration of 2e⁸cells/ml.

Loading Cells into Microfluidic Device

Splenocytes were imported into the microfluidic chip and loaded intopens containing 20-30 cells per pen. 100 microliters of media was flowedthrough the device at 1 μL/s to remove unwanted cells. Temperature wasset to 36° C., and culture media was perfused for 30 minutes at 0.1μL/sec.

Antigen Specificity Assay

Cell media containing 1:2500 goat anti-mouse F(ab′)2-Alexa 568 wasprepared.

100 μL of CD45 beads were re-suspend in 22 μL of the cell mediacontaining the 1:2500 dilution of goat anti-mouse F(ab′)2-Alexa 568.

The resuspended CD45 beads were next flowed into the main channel of themicrofluidic chip at a rate of 1 μL/sec until they were located adjacentto, but just outside the pens containing splenocytes. Fluid flow wasthen stopped.

The microfluidic chip was then imaged in bright field to determine thelocation of the beads.

Next, a Texas Red Filter was used to capture images of the cells andbeads. Images were taken every 5 minutes for 1 hr, with each exposurelasting 1000 ms and a gain of 5.

Results

Positive signal was observed developing on the beads, reflecting thediffusion of IgG-isotype antibodies diffusing out of certain pens andinto the main channel, where they were able to bind the CD45-coatedbeads. Binding of anti-CD45 antibody to the beads allowed for thesecondary goat anti-mouse IgG-568 to associate with the beads andproduce a detectable signal. See FIG. 5C, white arrows.

Using the methods disclosed herein, each group of splenocytes associatedwith positive signal could be separated and moved into new pens as asingle cell and reassayed. In this manner, single cells expressinganti-CD45 IgG antibodies could be detected.

Example 3. Membrane Preparation and Conjugation of Membrane Antigens toBeads

The following protocol demonstrates the feasibility of obtaining samplescontaining proteins (and other biomolecules), and particularly membranebound or membrane associated proteins, from samples cells of interest.The protocol is performed on Jurkat cells to enable testing of theresulting samples for proteins known to be present in such cells. Theprotocol could be readily extended to other cell types, including cancercells isolated from a tumor biopsy, thereby yielding samples containingtumor cell-specific (or other cell type-specific) antigens useful forscreening immunological cells (such as B cells) for their reactivity tothe antigens.

Materials:

-   -   Jurkat cells (ATCC TIB-152)    -   LiDS-Sample Buffer: 0.02 g/mL LiDS, 10% glycerol, 0.51 mM EDTA,        247 mM Tris, pH8.5 (ThermoFisher B0008)    -   Qiashredder (Qiagen 79654)    -   DPBS (containing calcium and magnesium, ThermoFisher 14040-182)    -   UltraPure Water (ThermoFisher 10977-015)    -   EZ-Link Sulfo-NHS-SS-Biotin (ThermoFisher 21328): store at −20°        C., take out and warm at RT 30 min before use; spin down briefly        to collect everything at the bottom of the tube; immediately        before use, add 164 μL ultrapure water to 1 tube containing 1 mg        Sulfo-NHS-SS-Biotin and pipette up and down several times to        dissolve (results in a 10 mM solution)    -   Complete Ultra mini (Sigma 5892791001): Prepare 20× solution:        dissolve 1 tablet in 500 μL water by vortexing (stable for 4        weeks at 4° C. or −20° C.)    -   Minute Plasma Membrane Protein Isolation Kit (Invent        Biotechnologies SM-005): Before use, thaw Solutions A and B, and        add protease inhibitors:        -   for each sample 500 μL Buffer A +25 μL 20× protease            inhibitors (store on ice)        -   for each sample 200 μL Buffer B+10 μL 20× protease inhibitor            (store on ice)        -   prepare 1 filter cartridge for each sample on ice    -   BSA: Chromatopur Bovine Albumin (MP Biomedicals 02180561)    -   Streptavidin coated magnetic microspheres (Bangs Laboratories        CM01N Lot #11806), wash and block before use:        -   Vortex beads, take out 300 μL/sample and transfer into 1.5            mL tube        -   Wash with PBS        -   Resuspend in 0.5% BSA and incubate at 4° C. (rotating) for            at least 1 h        -   Wash with PBS, resuspend in 500 μL DPBS (+ protease            inhibitors)/sample        -   Anti-alpha 1 Sodium Potassium ATPase (Na/K-ATPase) antibody            (Abcam ab7671)    -   Anti-human CD3 antibody, clone SK7 (BioLegend 344802)    -   Anti-human CD45 antibody, clone HI30 (BioLegend 304002)    -   F(ab′)2-Goat anti-Mouse IgG (H+L), Alexa Fluor 488 (ThermoFisher        A-11017)

Biotinylation of Membrane Proteins:

-   -   Count Jurkat cells    -   Prepare an aliquot for Western Blot analysis:        -   centrifuge 2×106 cells        -   wash with PBS        -   lyse in LiDS-Sample Buffer and centrifuge through a            Qiashredder column to shear DNA        -   store at 4° C. for a few days, longer at −80° C.    -   Wash multiples of 15×10⁶ cells three times with DPBS by        centrifugation    -   Resuspend each pellet in 500 μL DPBS, transfer into a 1.5 mL        tube and add 10 μL of freshly prepared 10 mM Sulfo-NHS-SS-Biotin    -   Rotate 30 min at RT    -   Wash three times with cold DPBS and proceed with cell pellets to        enrich the membrane fractions    -   Process 1 sample for Western Blot analysis (see above).

FIG. 6 shows an exemplary Western blot stained for Na/K-ATPase, aprotein present in the plasma membrane, and GAPDH, a cytosolic protein.

Enrichment of the Membrane Fraction using Minute Plasma MembraneIsolation Kit:

-   -   Perform all steps on ice, centrifuge at 4° C.    -   Resuspend pellets in Buffer A: 200 for <5×106 cells, 500        for >5×106 cells    -   Incubate on ice for 10 min    -   Vortex vigorously for 10-30 sec, immediately transfer to the        filter cartridge on ice    -   Cap the filter cartridge, centrifuge 30 sec at 16,000 g        (increase to 2 min if cell lysate does not go through)    -   Resuspend the pellet in collection tube, transfer to the same        filter and centrifuge again    -   Discard filter, resuspend pellet by vigorously vortexing for 10        sec    -   Centrifuge 1 min at 700 g (to pellet intact nuclei)    -   Centrifuge supernatant in a fresh 1.5 ml tube 30 min at 16,000        g/4° C. (→supernatant contains cytosol, pellet=total membrane        protein fraction)    -   Resuspend pellet in 200 μl buffer B by vortexing, or pipetting        up and down    -   Centrifuge 20 min at 7,800 g/4° C.→pellet contains organelle        membrane proteins    -   Carefully transfer supernatant (membrane fraction) into a fresh        2.0 ml tube

Bind Membrane Fractions to Streptavidin-Coated Magnetic Microspheres:

-   -   Add 80 μL of each membrane fraction to 500 μL beads    -   Rotate o/n at 4° C.    -   Save unbound fraction for analysis by Western Blot    -   Rotate 30 min in 0.5% BSA/PBS+ protease inhibitors    -   Wash with PBS    -   Resuspend in 250 μL 0.1% BSA/PBS+ protease inhibitors    -   Release Membrane Fractions from Beads (cleave S—S bond using        DTT):    -   Take out an aliquot of 50 μL beads    -   put on magnet and remove solution    -   Resuspend beads in 50 μL LiDS-Sample Buffer containing 50 mM DTT        (1× Reducing Agent, ThermoFisher B0009)    -   Incubate 2 hr at RT (mix every ˜30 min)

Antibody Staining:

-   -   Take 50 μL aliquots of beads (from step 24) for each antibody in        following dilutions        -   CD3 (1:200)        -   CD45 (1:100)        -   Na/K-ATPase (1:100)        -   No primary antibody    -   Rotate 30 min at 4° C.    -   Add 800 μL PBS to wash, wash 1× additionally with PBS    -   Incubate in secondary antibody (Alexa488-conjugated goat        anti-mouse) for 30 min at 4° C. (rotating, volume: 200 μL in        0.5% BSA/PBS+ protease inhibitors) 1:300    -   Add 800 μL PBS to wash, wash 1× additionally with PBS    -   Resuspend in 20 μL 0.5% BSA/PBS+ protease inhibitors    -   Image on fluorescence microscope

FIG. 7 shows beads coated with a Jurkat cell membrane fraction stainedfor CD3, CD45 or Na/K-ATPase. CD3 and CD45 are both strongly representedcompared with NA/K-ATPase, which appears as fainter spots. FIG. 8 showsa comparison with HEK293 cells. Here Na/K-ATPase stains the same, butCD3 and CD45 are absent.

The results demonstrate that proteins isolated from cell membranepreparations can be successfully coupled to beads. Such beads could bemixed with immunological cells to determine which cells are reactive toantigens (whether protein or otherwise) present in the cell membranepreparations.

Example 4. Identifying B Cells that Express Antibodies that Bind toBead-Conjugated Cancer Cell-Derived Antigens

A screen can be performed to identify patient-specific B cells thatexpress antibodies that bind to a patient's own medullary breast cancercells. The experimental design can include the following steps:

-   -   Harvest and selection of B cells and cancer cells;    -   Obtain cell membrane preparations from the cancer cells and        conjugate the membrane antigens to beads;    -   Load B cells into a microfluidic device;    -   Load beads conjugated to cancer cell-derived membrane antigens        into the microfluidic device; and    -   Assay for binding of antibodies produced by B cells to the        beads.

A. Cell Harvest and Selection

A biopsy of a medullary breast cancer tumor is obtained from a patientand the cells of the biopsy are dissociated, as described in Example 1(above). B cells are selected from the resulting dissociated cell sampleusing anti-CD-20 magnetic beads, thereby producing a B cell-enrichedcell sample. Cells remaining in the B cell-depleted dissociated cellsample are next depleted of non-medullary breast cancer cells using amixture of anti-ER (estrogen receptor), anti-PR (progesterone receptor),and anti-HER2 magnetic beads to produce a triple-negative breast cancercell-enriched sample.

B. Preparation of Cell Membrane from Breast Cancer Cells and Conjugationof the Membrane Antigens to Beads

Starting with the triple-negative breast cancer cell-enriched sampleobtained in part A, cell membranes from the cancer cells can be preparedand conjugated to beads, as described in 3, above.

C. Loading Cells into Microfluidic Device

The B cell-enriched cell sample is next loaded into a microfluidic chipby flowing 1-3 microliters of the sample into a flow channel at a rateof 0.1-1.0 microliters/sec. The flow of sample is then stopped and,while the cells in the sample are located in the flow channel, B cellsare selectively captured using DEP force (e.g., OET) and moved into theisolation regions of isolation chambers connected to the flow channel. Asingle B cell is placed into each of a plurality of isolation chambers.Depending on the size of the microfluidic chip used, 3000 or more Bcells can be individually isolated on a single chip. In general, fewer Bcells are expected to be present in the sample. As a result, all B cellsin the sample can be isolated. Once the B cells are moved into theisolation chambers, the flow channel is flushed with fresh medium toclear the flow channel of unwanted cells and debris.

After clearing the flow channel, the triple-negative breast cancercell-enriched sample is loaded into the chip. Similar to the Bcell-enriched sample, 1-3 microliters of the cancer cell-enriched sampleis flowed into the flow channel at a rate of 0.1-1.0 microliters/sec,then the flow is stopped. Using morphological traits, a plurality ofcancer cells (e.g., 3-6) is selected using DEP force (e.g., OET) andmoved into each isolation chamber that contains a B cell. Once thecancer cells are moved into the isolation chambers, the flow channel isflushed with fresh medium to clear the flow channel of unwanted cellsand debris. In this manner, a plurality of isolation chambers in themicrofluidic chip are loaded with a single B cell and a plurality ofmedullary breast cancer cells.

D. Loading Beads into the Microfluidic Device

After clearing the flow channel, the beads conjugated to the membraneantigens obtained from the triple-negative breast cancer cell-enrichedsample are loaded into the chip. Similar to the B cell-enriched sample,1-3 microliters of the cancer cell-associated antigen-conjugated beadsare flowed into the flow channel at a rate of 0.1-1.0 microliters/sec,then the flow is stopped. The beads are moved into each isolationchamber that contains a B cell. Once the beads are moved into theisolation chambers, the flow channel is subsequently flushed with freshmedium to clear the flow channel of unwanted beads and debris. In thismanner, a plurality of isolation chambers in the microfluidic chip areloaded with a single B cell and a plurality of cancer cell-associatedantigen-conjugated beads. Alternatively, the beads can be left in thechannel, as described in Example 2, above.

E. Antibody Binding Assay

To determine whether any of the isolated B cells are producingantibodies that bind to the cancer cell-associated antigens, the B cellsin the microfluidic chip are incubated under conditions conducive toantibody expression. After such incubation, a medium containing amixture of secondary antibodies that bind to the constant region ofhuman antibodies is flowed into the microfluidic chip, then stopped.Antibodies in the antibody mixture, which can contain fluorescentlylabeled anti-human IgG antibodies, anti-human IgM antibodies, andoptionally antibodies against human IgA, IgE, and/or IgD, are allowed todiffuse into the isolation chambers and bind to antibodies produced bythe B cells. If the antibodies produced by a B cell binds to one or moreof the cancer cell-associated antigens that are conjugated to the beadsin the corresponding isolation chamber, then the anti-human secondaryantibodies (and their labels) will become detectably bound to thesurface of the beads. If the beads are left in the channel, then thefluorescently labeled secondary antibodies can be mixed with the beadsbefore the beads are flowed into the microfluidic device.

During the secondary antibody incubation step, the microfluidic chip isperiodically imaged using a filter for detection of fluorescent signal(e.g., a Texas Red Filter). Images can be taken every 5 minutes for 1hr, with each exposure lasting 1000 ms.

F. Results

Positive signal can be observed developing on the surface of the beadsin (or proximal to) any isolation chamber that contains a B cell thatexpresses antibodies that bind to the cancer cell-associated antigens.Using this method, individual B cells expressing antibodies of interestcan be specifically identified. B cells that do not produce anyinteresting antibodies can be moved out of their isolation chambers andexported out of the microfluidic chip to waste. B cells identified asexpressing an antibody that binds to the patient's medullary breastcancer cell-derived antigens can be moved out of the isolation chamber(e.g., using DEP force, such as OET) and exported for amplification andsequencing of the heavy and light chain variable regions of theexpressed antibodies. If the B cells expanded while being induced toexpress antibodies, a clonal population of B cells can be exported foramplification and sequencing of the heavy and light chain variableregions of the expressed antibodies.

Example 5. Identifying B Cells that Express Antibodies that Bind to theCancer Cells

Alternatively, the screen can be performed to identify patient-specificB cells that express antibodies that bind to a patient's own medullarybreast cancer cells. The experimental design can include the followingsteps:

-   -   Harvest and selection of B cells and cancer cells;    -   Load cells into a microfluidic device; and    -   Assay for binding of antibodies produced by B cells to cancer        cells.

A. Cell Harvest and Selection

A biopsy of a medullary breast cancer tumor is obtained from a patientand the cells of the biopsy are dissociated, as described in Example 1(above). B cells are selected from the resulting dissociated cell sampleusing anti-CD-20 magnetic beads, thereby producing a B cell-enrichedcell sample. Cells remaining in the B cell-depleted dissociated cellsample are next depleted of non-medullary breast cancer cells using amixture of anti-ER (estrogen receptor), anti-PR (progesterone receptor),and anti-HER2 magnetic beads to produce a triple-negative breast cancercell-enriched sample.

B. Loading Cells into Microfluidic Device

The B cell-enriched cell sample is next loaded into a microfluidic chipby flowing 1-3 microliters of the sample into a flow channel at a rateof 0.1-1.0 microliters/sec. The flow of sample is then stopped and,while the cells in the sample are located in the flow channel, B cellsare selectively captured using DEP force (e.g., OET) and moved into theisolation regions of isolation chambers connected to the flow channel. Asingle B cell is placed into each of a plurality of isolation chambers.Depending on the size of the microfluidic chip used, 3000 or more Bcells can be individually isolated on a single chip. In general, fewer Bcells are expected to be present in the sample. As a result, all B cellsin the sample can be isolated. Once the B cells are moved into theisolation chambers, the flow channel is flushed with fresh medium toclear the flow channel of unwanted cells and debris.

After clearing the flow channel, the triple-negative breast cancercell-enriched sample is loaded into the chip. Similar to the Bcell-enriched sample, 1-3 microliters of the cancer cell-enriched sampleis flowed into the flow channel at a rate of 0.1-1.0 microliters/sec,then the flow is stopped. Using morphological traits, a plurality ofcancer cells (e.g., 3-6) is selected using DEP force (e.g., OET) andmoved into each isolation chamber that contains a B cell. Once thecancer cells are moved into the isolation chambers, the flow channel isflushed with fresh medium to clear the flow channel of unwanted cellsand debris. In this manner, a plurality of isolation chambers in themicrofluidic chip are loaded with a single B cell and a plurality ofmedullary breast cancer cells.

C. Antibody Binding Assay

To determine whether any of the isolated B cells are producingantibodies that bind to cancer cells, the B cells in the microfluidicchip are incubated under conditions conducive to antibody expression.After such incubation, a medium containing a mixture of secondaryantibodies that bind to the constant region of human antibodies isflowed into the microfluidic chip, then stopped. Antibodies in theantibody mixture, which can contain fluorescently labeled anti-human IgGantibodies, anti-human IgM antibodies, and optionally antibodies againsthuman IgA, IgE, and/or IgD, are allowed to diffuse into the isolationchambers and bind to antibodies produced by the B cells. If theantibodies produced by a B cell binds to one or more of the cancer cellsin the corresponding isolation chamber, then the anti-human secondaryantibodies (and their labels) will become detectably bound to thesurface of the medullary breast cancer cells.

During the secondary antibody incubation step, the microfluidic chip isperiodically imaged using a filter for detection of fluorescent signal(e.g., a Texas Red Filter). Images are taken every 5 minutes for 1 hr,with each exposure lasting 1000 ms.

D. Results

Positive signal can be observed developing on the surface of the cancercells in any isolation chamber that contains a B cell that expressesantibodies that bind to the cancer cells. Using this method, individualB cells expressing antibodies of interest can be specificallyidentified. B cells that do not produce any interesting antibodies canbe moved out of their isolation chambers and exported out of themicrofluidic chip to waste. Each B cell identified as expressing anantibody that binds to the patient's medullary breast cancer cells canbe moved out of the isolation chamber (e.g., using DEP force, such asOET) and exported for amplification and sequencing of the heavy andlight chain variable regions of the expressed antibodies. If the B cellsexpanded while being induced to express antibodies, a clonal populationof B cells can be exported for amplification and sequencing of the heavyand light chain variable regions of the expressed antibodies.

Example 6. Combination Therapy Using CAR-Ts and an Oncolytic Virus

Chimeric antigen receptor (CAR)-redirected T cells have the potential toprovide increased benefits in cancer treatment. A patient having cancer,such as neuroblastoma, undergoes a biopsy or surgical resection and Bcells producing antibodies that bind to the cancer are identified usingthe preceding examples. A CAR-T cell line is engineered to expressantibodies or fragments thereof corresponding to the antibodies producedby the identified B cell (by corresponding, we mean having at least thesame heavy chain CDRs, at least the same heavy and light chain CDRs, atleast the variable heavy chain, or at least the variable heavy and lightchain). For this patient, the CAR-T cell line is engineered to expressan scFv, where the genes coding the variable regions of the heavy chainand light chain are cloned by RT-PCR. Combinatorial scFv genes aregenerated by splicing-by-overlap PCR and then ligated into restrictionsites of a vector phage DNA.

The scFv sequence is cloned in frame into a retroviral backbone, such asthe SFG retroviral backbone. A retroviral supernatant is prepared using239T cells. Supernatant containing the retrovirus is collected 48 and 72hours later. For transduction, 0.5×10⁶/ml peripheral blood mononuclearcells (PBMCs) activated with OKT3 (Ortho Biotech, Bridgewater, N.J.) andCD28 (Becton Dickinson, Mountain View, Calif.) antibodies andrecombinant human IL-12 (100 units/ml; Proleukin, Chiron, Emeryville,Calif.) are plated in complete media (RPMI 1640 45%, Click medium 45%,and 10% heat-sterilized human serum and 2 mM L-glutamine) in 24-wellplates pre-coated with a recombinant fibronectin fragment (FN CH-296,Retronectin, Takara Shuzo, Otsu, Japan). After the addition of viralsupernatant, the cells are spun and incubated at 37° C. in 5% CO2. CARexpression on T lymphocytes is measured 72 hours later and the cellsmaintained in culture in complete media with the addition of rIL-2 (50units/ml) every 3 days.

Such CAR-T cells are adoptively transferred to the patient along withadministration of an oncolytic virus, such as an oncolytic adenovirus.The oncolytic adenovirus can be Ad5D24, optionally expressingrecombinant IL15, RANTES, or the combination thereof. The patient isexpected to have significant clinical improvement.

LISTING OF EXEMPLARY EMBODIMENTS

1. A method of preparing an antibody therapeutic, the method comprising:providing a dissociated cell sample from at least one solid tumor sampleobtained from a patient; loading the dissociated cell sample into amicrofluidic device having at least one flow region and at least oneisolation region fluidically connected to the flow region; moving atleast one B cell from the dissociated cell sample into at least oneisolation region in the microfluidic device, thereby obtaining at leastone isolated B cell; and identifying at least one isolated B cell thatproduces antibodies capable of binding to a cancer cell-associatedantigen.

2. The method of embodiment 1, wherein said isolation region comprisesat least one conditioned surface that promotes B cell lymphocyteviability, said conditioned surface comprising covalently linkedmolecules.

3. The method of embodiment 1, wherein said isolation region comprises aplurality of conditioned surfaces that promote B cell lymphocyteviability, each conditioned surface comprising covalently linkedmolecules.

4. The method of embodiment 2 or 3, wherein said at least oneconditioned surface or each conditioned surface of said pluralitycomprises a layer of covalently linked hydrophilic molecules.

5. The method of embodiment 4, wherein said hydrophilic molecules areselected from the group of polymers comprising polyethylene glycol(PEG), polymers comprising amino acids, and combinations thereof.

6. The method of embodiment 1, wherein said isolation region comprisesat least one surface coated with a coating material that promotes B cellviability.

7. The method of embodiment 1, wherein said isolation region comprises aplurality of surfaces each coated with a coating material that promotesB cell viability.

8. The method of embodiment 6 or 7, wherein said coating materialcomprises hydrophilic molecules.

9. The method of embodiment 8, wherein said hydrophilic molecules areselected from the group of polymers comprising polyethylene glycol(PEG), polymers comprising amino acids, and combinations thereof.

10. The method of any one of embodiments 1 to 9, wherein each of the atleast one isolation region(s) forms a dead-end in the microfluidicdevice, and wherein, when the flow region is substantially filled with aflowing first fluidic medium and the isolation region(s) aresubstantially filled with a second fluidic medium: components of thesecond medium are able to diffuse into the first medium and componentsof the first medium are able to diffuse into the second medium; andthere is substantially no flow of the first medium from the flow regioninto the isolation region.

11. The method of any one of embodiments 1 to 10, wherein the flowregion of the microfluidic device comprises a microfluidic channel.

12. The method of embodiment 11, wherein each of the at least oneisolation regions is part of a corresponding sequestration chamber, andwherein each sequestration chamber further comprises a connection regionfluidically connecting the corresponding isolation region to themicrofluidic channel.

13. The method of embodiment 12, wherein the connection region has awidth W_(con) of about 20 microns to about 60 microns.

14. The method of embodiment 12 or 13, wherein each of the at least oneisolation region(s) has a width W_(iso) that is greater than the widthW_(con) of the corresponding connection region.

15. The method of embodiment 14, wherein each of the at least oneisolation region(s) has a width W_(iso) that is about 50 microns toabout 250 microns.

16. The method of any one of embodiments 12 to 15, wherein each of thesequestration chambers comprises a volume of about 0.5 nl to about 2.5nl.

17. The method of any one of embodiments 1 to 16, wherein the at leastone B cell is moved into the at least one isolation region using gravityand/or localized fluid flow.

18. The method of any one of embodiments 1 to 17, wherein themicrofluidic device comprises a substrate having a DEP configuration.

19. The method of embodiment 18, wherein moving the at least one B cellinto the at least one isolation region comprises using DEP force to movethe at least one B cell.

20. The method of any one of embodiments 1 to 19, wherein prior toloading the dissociated cell sample, the method further compriseslabeling B cells in the dissociated cell sample with a detectablemarker.

21. The method of embodiment 20, wherein moving the at least one B cellinto the at least one isolation region comprises selecting at least oneB cell for movement based on detection of the detectable marker.

22. The method of any one of embodiments 1 to 21, wherein moving atleast one B cell into at least one isolation region comprises moving aplurality of individual B cells into a corresponding plurality ofseparate isolation regions.

23. The method of any one of embodiments 1 to 22, further comprising:contacting the at least one B cell with a stimulating agent thatstimulates B cell activation.

24. The method of embodiment 23, wherein the stimulating agent comprisesa CD40 agonist.

25. The method of embodiment 24, wherein the CD40 agonist comprisesCD40L, a derivative thereof, or an anti-CD40 antibody.

26. The method of embodiment 23, wherein the stimulating agent comprisesone or more CD40L+ feeder cells.

27. The method of embodiment 26, wherein the one or more CD40L+ feedercells is/are T cells or a derivative thereof.

28. The method of any one of embodiments 23 to 27, wherein thestimulating agent comprises a toll-like receptor (TLR) agonist.

29. The method of embodiment 28, wherein the TLR agonist is a CpGoligonucleotide.

30. The method of any one of embodiments 23 to 29, wherein the at leastone B cell is contacted with the stimulating agent for a period of oneto ten days.

31. The method of embodiment 30, wherein the at least one B cell iscontacted with the stimulating agent substantially continuously for saidperiod of one to ten days.

32. The method of any one of embodiments 23 to 31, further comprising:providing culture medium to the at least one B cell, wherein the culturemedium comprises one or more growth-inducing agents that promote B cellexpansion.

33. The method of embodiment 32, wherein the one or more growth-inducingagents include at least one agent selected from the group of IL-2, IL-4,IL-6, IL-10, IL-21, and BAFF.

34. The method of embodiment 32 or 33, wherein the provided culturemedium comprises the stimulating agent.

35. The method of any one of embodiments 32 to 34, wherein the at leastone B cell is provided culture medium for a period of one to ten days.

36. The method of any one of embodiments 32 to 35, wherein each B cellof the at least one isolated B cell(s) is cultured in the isolationregion of the microfluidic device to a cell count of about 8 to 20cells.

37. The method of any one of embodiments 32 to 36, wherein the steps ofcontacting the at least one B cell with the stimulating agent andproviding culture medium to the at least one B cell are preformed over asubstantially coextensive period of time.

38. The method of any one of embodiments 23 to 37, wherein contactingthe at least one B cell with the stimulating agent is performed prior toloading the dissociated cell sample into the microfluidic device.

39. The method of any one of embodiments 23 to 38, wherein contactingthe at least one B cell with the stimulating agent is performed aftermoving the at least one B cell into the at least one isolation region.

40. The method of any one of embodiments 23 to 39, wherein contactingthe at least one B cell with the stimulating agent is performed duringthe step of identifying the least one isolated B cell that producesantibodies capable of binding to the cancer cell-associated antigen.

41. The method of any one of embodiments 1 to 40, wherein identifyingthe at least one isolated B cell that produces antibodies capable ofbinding to the cancer cell-associated antigen comprises introducing thecancer cell-associated antigen into the microfluidic device.

42. The method of embodiment 41, wherein introducing the cancercell-associated antigen into the microfluidic device comprisesintroducing a fluidic medium comprising the cancer cell-associatedantigen into the microfluidic device.

43. The method of embodiment 42, wherein the cancer cell-associatedantigen is solubilized in the fluidic medium.

44. The method of embodiment 41, wherein introducing the cancercell-associated antigen into the microfluidic device comprisesintroducing micro-objects into the microfluidic device, wherein themicro-objects comprise the cancer cell-associated antigen.

45. The method of embodiment 44, wherein the micro-objects are selectedfrom cells, liposomes, lipid nanorafts, and beads.

46. The method of embodiment 45, wherein the micro-objects are beads,and wherein the cancer cell-associated antigen is conjugated to thebeads.

47. The method of embodiment 45 or 46, wherein the cancercell-associated antigen is a membrane-associated antigen present on thecell surface of cancer cells present in the at least one tumor sample.

48. The method of embodiment 47, wherein the cancer cell-associatedantigen conjugated to the beads is from a cell membrane preparationobtained from cancer cells isolated from the at least one tumor sample.

49. The method of embodiment 45, wherein the micro-objects are cancercells.

50. The method of embodiment 49, wherein the cancer cells exhibitmarkers that are exhibited by cancer cells from the at least one tumorsample obtained from the patient.

51. The method of embodiment 49, wherein the cancer cells are isoloatedfrom the at least one tumor sample.

52. The method of embodiment 49 or 50, wherein the cancer cells areisolated from one or more tumor samples obtained from a differentpatient.

53. The method of embodiment 52, wherein the patient from which the atleast one solid tumor sample was obtained and the different patient havebeen diagnosed with a same type of cancer.

54. The method of embodiment 49 or 50, wherein the cancer cells are froma cancer cell line.

55. The method of any one of embodiments 41 to 54, wherein introducingthe cancer cell-associated antigen into the microfluidic devicecomprises: flowing a fluidic medium that contains micro-objects thatcomprise the cancer cell-associated antigen through the flow region ofthe microfluidic device; and stopping the flow of the fluidic mediumwhen at least some of the micro-objects in the medium are located in aportion of the flow region that is proximal to the at least oneisolation region.

56. The method of embodiment 54, wherein introducing the cancercell-associated antigen into the microfluidic device further comprisesmoving at least one of the micro-objects into at least one isolationregion in the microfluidic device.

57. The method of embodiment 56, wherein moving the at least onemicro-object into the at least one isolation region in the microfluidicdevice comprises moving at least one micro-object into each of aplurality of isolation regions.

58. The method of embodiment 56 or 57, wherein the at least onemicro-object is moved into at least one isolation region that containsat least one B cell, thereby producing at least one isolation regionhaving at least one micro-object and at least one B cell.

59. The method of embodiment 56, wherein the at least one micro-objectand the at least one B cell are moved into different isolation regions.

60. The method of embodiment 59, wherein the different isolation regionsare adjacent to one another within the microfluidic device.

61. The method of any one of embodiments 41 to 60, wherein identifyingthe at least one isolated B cell that produces antibodies capable ofbinding to a cancer cell-associated antigen further comprises: flowing afluidic medium that contains a labeled antibody-binding agent throughthe flow region of the microfluidic device; stopping the flow of thefluidic medium when at least some of the labeled antibody-binding agentin the fluidic medium is located in a portion of the flow region that isproximal to the at least one isolation region; and monitoring binding ofthe labeled antibody-binding agent to the cancer cell-associatedantigen.

62. The method of embodiment 61, wherein the labeled antibody-bindingagent is a labeled anti-IgG antibody.

63. The method of embodiment 61 or 62, wherein the labeledantibody-binding agent is covalently bound to a fluorescent label.

64. The method of any one of embodiments 61 to 63, wherein the labeledantibody-binding agent is provided in a mixture with the cancercell-associated antigen.

65. The method of any one of embodiments 61 to 63, wherein the labeledantibody-binding agent is provided after providing the cancercell-associated antigen.

66. The method of any one of embodiments 61 to 65, wherein monitoringbinding of the labeled antibody-binding agent to the cancercell-associated antigen comprises imaging the microfluidic device.

67. The method of embodiment 66, wherein the imaging comprisesfluorescence imaging.

68. The method of embodiment 66 or 67, wherein the imaging comprisestaking a plurality of images.

69. The method of embodiment 68, wherein the plurality of images aretaken at fixed time intervals.

70. The method of any one of embodiments 1 to 69, wherein thedissociated cell sample is obtained from one solid tumor sample.

71. The method of any one of esmbodiment 1 to 69, wherein thedissociated cell sample is obtained from multiple solid tumor samples.

72. The method of any one of embodiments 1 to 71, wherein providing adissociated cell sample comprises obtaining the at least one solid tumorsample, and dissociating single cells from the at least one solid tumorsample.

73. The method of any one of embodiments 1 to 72, wherein individualcells of the dissociated cell sample are dissociated from the at leastone tumor sample by: collagenase plus DNase digestion; and/or a celldissociator instrument.

74. The method of any of embodiments 1 to 72, wherein the step ofloading the dissociated cell sample comprises: fractionating thedissociated cell sample to isolate a B cell-enriched fraction that has agreater concentration of B cells than the original dissociated sample;and loading the B cell-enriched fraction into the microfluidic device.

75. The method of embodiment 74, wherein the fractionating comprisesselecting B cells from the dissociated cell sample using at least onemarker chosen from CD19, CD20, IgM, IgD, CD38, CD27, CD138, PNA, andGL7.

76. The method of any one of embodiments 1 to 75, wherein prior toloading the dissociated cell sample or the B cell-enriched fraction intothe microfluidic device, the method further comprises labeling cancercells in the dissociated cell sample with a detectable marker.

77. The method of any of embodiments 1 to 76, wherein the step ofloading the dissociated cell sample further comprises: fractionating thedissociated cell sample to isolate a cancer cell-enriched fraction thathas a greater concentration of cancer cells than the originaldissociated sample; and loading the cancer cell-enriched fraction intothe microfluidic device.

78. The method of embodiment 76, wherein the method further comprisesselecting at least one cancer cell for movement into at least oneisolation region.

79. The method of embodiment 77, wherein the method further comprisesselecting at least one cancer cell for movement into at least oneisolation region, and wherein the at least one cancer cell is selectedbased on detection of the detectable marker.

80. The method of embodiment 79, wherein the at least one cancer cellsis loaded into the microfluidic device and moved into the isolationregion of the microfluidic device before the B cells are loaded into themicrofluidic device and moved into the isolation regions.

81. The method of embodiment 77, wherein the cancer cell-enrichedfraction is loaded into the flow region of the microfluidic device afterthe B cells are loaded into the microfluidic device and moved into theisolation regions.

82. The method of embodiment 77, wherein the cancer cells do not enterthe isolation regions.

83. The method of any one of embodiments 76 to 82, wherein the cancercells are selected from the dissociated cell sample or enriched using atleast one marker specific for the cancer (e.g., any of thecancer-associated markers disclosed herein).

84. The method of any one of embodiments 76 to 83, wherein the cancercells are separated from the dissociated cell sample or enriched usingmorphological differences.

85. The method of any one of embodiments 76 to 84, wherein the cancercells are selected from the dissociated cell sample using at least onemarker that is down-regulated in cancer cells, to isolate normal cellsaway from the cancer cells.

86. The method of any one of embodiments 74 to 85, wherein the B cellsand/or cancer cells are selected from the dissociated cell sample byFACS.

87. The method of any one of embodiments 74 to 86, wherein the B cellsand/or cancer cells are selected from the dissociated cell sample bymagnetic bead-based sorting.

88. The method of any one of embodiments 1 to 87, wherein the solidtumor sample is a tumor biopsy.

89. The method of any one of embodiments 1 to 88, wherein the tumor thatyielded the at least one tumor sample has a tertiary lymphoid structure.

90. The method of any one of embodiments 1 to 89, wherein the tumor thatyielded the at least one tumor sample is a breast cancer, genitourinarycancer (such as a cancer originating in the urinary tract, such as inthe kidneys (e.g., renal cell carcinoma), ureters, bladder, or urethra;cancer of the male reproductive tract (e.g., testicular cancer, prostatecancer, or a cancer of the seminal vesicles, seminal ducts, or penis);or of the female reproductive tract (e.g., ovarian cancer, uterinecancer, cervical cancer, vaginal cancer, or a cancer of the fallopiantubes)), a cancer of the nervous system (such as neuroblastoma),intestinal cancer (such as colorectal cancer), lung cancer, melanoma, oranother type of cancer.

91. The method of embodiment 90, wherein the tumor is a medullary breastcancer.

92. The method of embodiment 90, wherein the tumor is a mesothelioma.

93. The method of any one of embodiments 1 to 92, wherein the methodfurther comprises: exporting the at least one identified B cell, or apopulation of cloned B cells derived therefrom, from the microfluidicdevice.

94. The method of embodiment 93, wherein each identified B cell, or thepopulation of cloned B cells derived therefrom, is exportedindividually.

95. The method of any one of embodiments 1 to 94, further comprising:performing antibody sequencing on the at least one identified B cell, oron some or all of a clonal population of B cells derived therefrom.

96. The method of embodiment 95, wherein performing antibody sequencingcomprises determining paired heavy chain and light chain variable domainantibody sequences from the at least one identified B cell, or from someor all of the clonal population of B cells derived therefrom.

97. The method of any one of embodiments 1 to 96, wherein the methodfurther comprises generating an antibody therapeutic comprising some orall of the paired heavy chain and light chain variable domain sequencesfrom the at least one identified B cell.

98. The method of any one of embodiments 1 to 97, wherein T or NK cellsare present in the dissociated cell sample.

99. The method of embodiment 98, wherein the method further comprisesperforming a selection on the dissociated cell sample to isolate afraction that has a greater concentration of T or NK cells than theoriginal dissociated sample.

100. The method of embodiment 98 or 99, wherein the T or NK cells areobtained from the same patient's solid tumor sample(s).

101. The method of any one of embodiments 98 to 100, wherein the T cellsare separated from the dissociated cell sample by using at least onemarker chosen from CD4, CD8, CD25, CD45RA, CD45RO, CD62L, CD69, and CD3;or wherein the NK cells are separated from the dissociated cell sampleby using CD56 as a marker.

102. The method of any one of embodiments 98 to 101, wherein individualT or NK cells are selected and cloned from the patient.

103. The method of any one of embodiments 1 to 102, wherein the methodcomprises using a microfluidic device to identify at least one B cellthat produces antibodies capable of binding to both the patient's cancercells and cancer cells from another source.

104. The method of any one of embodiments 1 to 103, wherein the methodfurther comprises using a microfluidic device to identify whether theantibodies produced by the B cell(s) are capable of binding tonon-cancer cells.

105. The method of embodiment 104, wherein the non-cancer cells and thecancer cells are from the patient that provided the at least one tumorsample.

106. The method of embodiment 104 or 105, wherein the same microfluidicdevice is used to identify binding of the antibodies to cancer andnon-cancer cells.

107. The method of embodiment 106, wherein the cancer and non-cancercells are loaded sequentially in the same flow path.

108. A method of treating a patient having cancer, the method comprisingtreating the patient with an antibody or fragment thereof produced bythe method of any one of embodiments 1 to 107.

109. The method of embodiment 108, wherein the tumor sample is takenfrom the same patient who is treated.

110. The method of embodiment 108 or 109, wherein the time fromobtaining the tumor sample to treatment is at most about 2 months.

111. The method of any one of embodiments 108 to 110, wherein theantibody or fragment thereof is a single chain antibody.

112. The method of any one of embodiments 108 to 110, wherein theantibody or fragment thereof has two heavy chains and two light chains.

113. The method of any one of embodiments 108 to 111, wherein theantibody is displayed on an engineered T or NK cell.

114. The method of embodiment 113, wherein the engineered T cell is achimeric antigen receptor T cell or the engineered NK cell is a chimericantigen receptor NK cell.

115. The method of embodiment 113 or 114, wherein the T or NK cell wasobtained from the same patient prior to being engineered to display theantibody.

116. The method of embodiment 115, wherein the T or NK cell was obtainedfrom the patient's tumor sample.

117. The method of any one of embodiments 108 to 116, wherein theantibody or fragment thereof is administered in combination with anothertherapy.

118. The method of claim 117, wherein the combination therapy issurgery, radiation, chemotherapy, CAR-T cell therapy, CAR-NK celltherapy, T cell therapy, other immunotherapy, or administration ofimmune-stimulatory molecule or a tumor-specific virus.

119. A method of labeling or detecting cancer in a patient comprisingadministering an antibody or fragment thereof conjugated to a detectablelabel, wherein the antibody or fragment thereof is produced by themethod of any one of embodiments 1 to 107.

120. An engineered antibody construct comprising: at least the heavychain CDRs of an antibody identified by the method of any one ofembodiments 1 to 107; at least the light chain CDRs of an antibodyidentified by the method of any one of embodiments 1 to 107; at leastthe heavy and light chain CDRs of an antibody identified by the methodof any one of embodiments 1 to 107; at least the heavy chain variableregion of an antibody identified by the method of any one of embodiments1 to 107; at least the light chain variable region of an antibodyidentified by the method of any one of embodiments 1 to 107; or at leastthe heavy and light chain variable regions of an antibody identified bythe method of any one of embodiments 1 to 107.

121. An engineered antibody construct, wherein the engineered antibodyconstruct is a variant of the antibody identified by the method of anyone of embodiments 1 to 107 (e.g., a variant having 1 to 20, 1 to 15, 1to 10, or 1 to 5 amino acid substitutions, additions, or deletions inthe light chain and/or heavy chain variable regions of the identifiedantibody).

122. The engineered antibody construct of embodiment 120 or 121, whereinthe construct is a Fab, Fab′(2), scFv, multivalent scFv, minibody,bispecific antibody, or camel variable functional heavy chain domain.

123. An engineered T or NK cell comprising an antibody or fragmentthereof displayed on its external surface, wherein the antibody orfragment thereof is identified by the method of any one of embodiments 1to 107.

124. The engineered T or NK cell of embodiment 123, wherein the T or NKcell and the antibody or fragment thereof were obtained from the samepatient.

125. A method of preparing an engineered T cell comprising an antibodyor fragment thereof displayed on its external surface, the methodcomprising: identifying an antibody that binds to a tumor sampleaccording to the method of any one of embodiments 1 to 107; andgenetically engineering a T cell to express the antibody or a fragmentthereof.

126. The method of embodiment 125, wherein the T cell, the B cellexpressing the antibody, and the tumor sample were obtained from thesame patient.

127. A method of preparing an engineered NK cell comprising an antibodyor fragment thereof displayed on its external surface, the methodcomprising: identifying an antibody that binds to a tumor sampleaccording to the method of any one of embodiments 1 to 107; andgenetically engineering a NK cell to express the antibody or a fragmentthereof.

128. The method of embodiment 127, wherein the NK cell, the B cellexpressing the antibody, and the tumor sample were obtained from thesame patient.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the embodiments. The foregoingdescription and Examples detail certain embodiments and describes thebest mode contemplated. It will be appreciated, however, that no matterhow detailed the foregoing may appear in text, the embodiment may bepracticed in many ways and should be construed in accordance with theappended claims and any equivalents thereof.

1. A method of identifying an isolated B cell that produces antibodiescapable of binding to a cancer cell-associated antigen comprising:loading a dissociated cell sample from at least one solid tumor sampleobtained from a patient into a microfluidic device having at least oneflow region and at least one sequestration chamber comprising anisolation region, wherein the isolation region is fluidically connectedto the flow region; moving at least one B cell from the dissociated cellsample into at least one isolation region in the microfluidic device,thereby obtaining at least one isolated B cell; and identifying at leastone isolated B cell that produces antibodies capable of binding to acancer cell-associated antigen. 2-17. (canceled)
 18. The method of claim1, wherein moving the at least one B cell into the at least oneisolation region comprises using DEP force to move the at least one Bcell.
 19. The method of claim 1, wherein the at least one B cell ismoved into the at least one isolation region using gravity and/orlocalized fluid flow. 20-22. (canceled)
 23. The method of claim 1,further comprising: contacting the at least one B cell with astimulating agent that stimulates B cell activation.
 24. (canceled) 25.(canceled)
 26. The method of claim 23, wherein the stimulating agentcomprises one or more CD40L+ feeder cells.
 27. The method of claim 26,wherein the one or more CD40L+ feeder cells is/are T cells or aderivative thereof.
 28. (canceled)
 29. The method of claim 23, whereinthe stimulating agent further comprises a CpG oligonucleotide.
 30. Themethod of claim 23, wherein the at least one B cell is contacted withthe stimulating agent for a period of one to ten days.
 31. The method ofclaim 30, wherein the at least one B cell is contacted with thestimulating agent substantially continuously for said period of one toten days.
 32. The method of claim 23, further comprising: providingculture medium to the at least one B cell, wherein the culture mediumcomprises one or more growth-inducing agents that promote B cellexpansion.
 33. (canceled)
 34. (canceled)
 35. The method of claim 32,wherein the at least one B cell is provided culture medium for a periodof one to ten days.
 36. (canceled)
 37. The method of claim 32, whereinthe steps of contacting the at least one B cell with the stimulatingagent and providing culture medium to the at least one B cell arepreformed over a substantially coextensive period of time.
 38. Themethod of claim 23, wherein contacting the at least one B cell with thestimulating agent is performed at least prior to loading the dissociatedcell sample into the microfluidic device.
 39. The method of claim 23,wherein contacting the at least one B cell with the stimulating agent isperformed at least after moving the at least one B cell into the atleast one isolation region.
 40. The method of claim 23, whereincontacting the at least one B cell with the stimulating agent isperformed at least during the step of identifying the least one isolatedB cell that produces antibodies capable of binding to the cancercell-associated antigen.
 41. The method of claim 1, wherein identifyingthe at least one isolated B cell that produces antibodies capable ofbinding to the cancer cell-associated antigen comprises introducing thecancer cell-associated antigen into the microfluidic device. 42-44.(canceled)
 45. The method of claim 44, wherein the micro-objects areselected from cells, liposomes, lipid nanorafts, and beads. 46-48.(canceled)
 49. The method of claim 45, wherein the micro-objects arecancer cells.
 50. The method of claim 49, wherein the cancer cellsexhibit markers that are exhibited by cancer cells from the patient's atleast one tumor sample.
 51. The method of claim 49, wherein the cancercells are isolated from the at least one tumor sample.
 52. The method ofclaim 50, wherein the cancer cells are isolated from one or more tumorsamples obtained from a different patient.
 53. (canceled)
 54. The methodof claim 49, wherein the cancer cells are from a cancer cell line.55-72. (canceled)
 73. The method of claim 1, wherein individual cells ofthe dissociated cell sample are dissociated from the at least one tumorsample by: a. collagenase plus DNase digestion; and/or b. a celldissociator instrument. 74-128. (canceled)